[Show abstract][Hide abstract] ABSTRACT: Non-steroidal anti-inflammatory drugs (NSAIDs) are cyclooxygenases (COXs) inhibitors frequently used in the treatment of acute and chronic inflammation. Side effects of NSAIDs are often due to their ability to induce apoptosis. Located at the Endoplasmic Reticulum membranes a tripartite signalling pathway, collectively known as the Unfolded Protein Response (UPR), decides survival or death of cells exposed to cytotoxic agents. To shed light on the molecular events responsible for the cytotoxicity of NSAIDs, we analysed the ability of diclofenac and indomethacin to activate the UPR in the human hepatoma cell line Huh7. We report that both NSAIDs can induce differently the single arms of the UPR. We show that indomethacin turns on the PERK and, only in part, the ATF6 and IRE1 pathways. Instead, diclofenac reduces the expression of ATF6 and does not stimulate the IRE1 endonuclease, which drives the expression of the prosurvival factor XBP1. Diclofenac, as well as indomethacin, is able to activate efficiently only the PERK pathway of the UPR, which induces the expression of the proapoptotic GADD153/CHOP protein. Our results highlight the importance of the UPR in evaluating the potential of drugs to induce apoptosis.
Full-text · Article · Sep 2011 · The Open Biochemistry Journal
[Show abstract][Hide abstract] ABSTRACT: Several diseases are characterized by chronic inflammation, a condition frequently associated with angiogenesis and fibrogenesis that account for the development of granulation tissue. Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) is a crucial modulator of intracellular prosurvival signaling pathways and is implicated in the pathogenesis of inflammatory process. In this study, we have investigated the role of NF-kappaB in the angiogenic and fibrogenic response induced by lambda-carrageenin in a rat model of chronic inflammation at 1, 3, and 5 days. The subcutaneous implant of lambda-carrageenin-soaked sponges in rat induced a time-related increase of granulation tissue formation accompanied by intense neovascularization. These lambda-carrageenin-induced changes were significantly reduced by coinjection of wild-type oligodeoxynucleotide (WT ODN) decoy to NF-kappaB. Molecular, morphological, and ultrastructural analysis performed on whole granulation tissue demonstrated: (1) inhibition of NF-kappaB/DNA binding activity; (2) downregulation of cyclooxygenase-2, matrix metalloproteinase-9, tumor necrosis factor-alpha, and vascular endothelial growth factor; (3) upregulation of thrombospondin (TSP)-1 at 1 day and TSP-2 at 5 days; and (4) increase in Bax to Bcl-2 ratio. Our findings show that the blockade of NF-kappaB activation by WT ODN decoy prevents the development of granulation tissue induced by lambda-carrageenin-soaked sponge implant upregulating Bax as well as TSP-1 and TSP-2 expression.
Full-text · Article · Mar 2009 · Journal of Molecular Medicine
[Show abstract][Hide abstract] ABSTRACT: Epidemiologic data demonstrate a protective role by normal androgen levels on cardiovascular health and erectile function. Low androgen levels are associated with erectile dysfunction and increased risk of cardiovascular diseases. Both conditions recognize as anatomic substrate a pathologic structural remodeling. Direct androgen effects on male external genitalia, vascular wall, and myocardium have been reported.
To review current knowledge about androgen-dependent molecular signaling pathways and cellular events within penile and cardiovascular tissues involved in the homeostatic control of morphologic tissue properties and in the development of structural remodeling in presence of normal and low androgen levels, respectively.
A literature search was performed in November 2008 using the commercially available Medline online engine search to retrieve studies (from 1998 to 2008) on the mechanisms mediating the role of androgens on penile and cardiovascular morphologic homeostasis and remodeling. A combination of the following medical subject headings was used: androgens, hypogonadism, vessel tissue architecture, remodeling, cardiovascular system, and penis.
Androgens exert direct beneficial effects on both cardiovascular and penile tissues. Endothelial cells and smooth-muscle cells are the main cellular targets for direct androgen effects in both tissues and are involved in pathologic remodeling in hypogonadal models. At vascular level, androgens promote endothelial cell survival, reduce endothelial expression of proinflammatory markers, and inhibit proliferation and intimal migration of vascular smooth-muscle cells. At penile level, low androgen levels are associated with apoptosis of endothelial cells and smooth-muscle cells. Moreover, low androgen levels impair proliferation, migration, and homing of endothelial progenitor cells as well as myogenic differentiation of mesenchymal progenitor cells.
Normal androgen levels promote vascular and penile homeostasis by direct mechanisms mainly involving endothelial cells and smooth-muscle cells. Low androgen levels are associated with impairments of such mechanisms, leading to pathologic structural remodeling.
No preview · Article · Feb 2009 · European Urology
[Show abstract][Hide abstract] ABSTRACT: To assess the efficacy of a comprehensive motivational approach in reducing blood pressure in Italian patients with hypertension.
Two hundred and ninety-two first visit patients with hypertension without diabetes and dyslipidemia and with BMI < 28 were enrolled. One hundred and forty-two were randomly assigned to a Control group (C) and 150 to an Intervention group (I). A ten-multiple-choice questionnaire was developed to evaluate the effect of the intervention on lifestyle modification. Patients were given the questionnaire, had their BP measured and drug therapy registered before educational intervention and 12 months later. Group I patients participated in the focus group and in the role play 2 and 4 months, respectively, after recruitment. Group C patients received the oral information.
Blood pressure values and lifestyle modification. An intention to treat analysis was undertaken. Analysis was performed using SPSS version 15.0.
Of the 150 group I patients, 58 participated in both focus group and role play, 30 participated only in focus group and the remaining 62 never participated. After 12 months, there was a significant reduction of BP for group I (P < 0.001) and a significant reduction only for systolic BP in group C (P = 0.01). Diastolic BP and systolic BP decreased more markedly in group I than in group C, with P < 0.001 for both. We found a significant improvement of lifestyle modification after 12 months of follow-up concerning some aspects in both groups.
Our findings show that a motivational approach is a powerful tool for achieving better blood pressure control and is an essential skill for all healthcare professionals.
No preview · Article · Jun 2008 · International Journal of Clinical Pharmacy
[Show abstract][Hide abstract] ABSTRACT: Serum deprivation induced in human lymphoblastoid Raji cells oxidative stress-associated apoptotic death and G0/G1 cell cycle arrest. Addition into culture medium of the immunomodulatory protein Seminal vesicle protein 4 (SV-IV) protected these cells against apoptosis but not against cycle arrest. The antiapoptotic activity was related to: (1) decrease of endocellular reactive Oxygen species (ROS) (2) increase of mRNAs encoding anti-oxidant enzymes (catalase, G6PD) and antiapoptotic proteins (survivin, cox-1, Hsp70, c-Fos); (3) decrease of mRNAs encoding proapoptotic proteins (c-myc, Bax, caspase-3, Apaf-1). The biochemical changes underlaying these effects were probably induced by a protein tyrosine kinase (PTK) activity triggered by the binding of SV-IV to its putative plasma membrane receptors. The ineffectiveness of SV-IV to abrogate the cycle arrest was accounted for by its downregulating effects on D1,3/E G1-cyclins and CdK2/4 gene expression, ppRb/pRb ratio, and intracellular ROS concentration. In conclusion, these experiments: (1) prove that SV-IV acts as a cell survival factor; (2) suggest the involvement of a PTK in SV-IV signaling; (3) point to cell cycle-linked enzyme inhibition as responsible for cycle arrest; (4) provide a model to dissect the cycle arrest and apoptosis induced by serum withdrawal; (5) imply a possible role of SV-IV in the survival of hemiallogenic implanting embryos.
[Show abstract][Hide abstract] ABSTRACT: The endoplasmic reticulum Golgi intermediate compartment 53 protein recycles continuously between the endoplasmic reticulum and the Golgi complex and ensures the anterograde transport of specific glycoproteins with the assistance of the Multiple Clotting Factor Deficiency adaptor protein. Therefore, to analyze the effect of the endoplasmic reticulum stress on the secretory pathway beyond the endoplasmic reticulum, we analyzed the expression of both proteins in J774 macrophages incubated with the nitric oxide donor DETA NONOate or with thapsigargin. Both proteins accumulated progressively, by a transcriptional mechanism, in response to these inducers. Nitric oxide also induced a higher level of calreticulin and glucose regulated 78 protein, two endoplasmic reticulum proteins controlled by the unfolded protein response. Interestingly, nitric oxide induced the processing of the activating transcription factor 6alpha of the unfolded protein response, while thapsigargin also induced the activation of the transcription factor X-box Binding Protein 1. In addition, we showed that the accumulation of both transporters occurred simultaneously with the activation of endoplasmic reticulum-stress-dependent apoptosis, suggesting that these proteins may participate in the events that will eventually decide the fate of the cell. Induction of endoplasmic reticulum stress affected the rate of anterograde transport of a reporter glycoprotein, indicating that the endoplasmic reticulum to Golgi transport is remarkably impaired. Our results indicate that increased levels of cargo receptor proteins might have a function either in the quality control of protein folding in the endoplasmic reticulum or in the homeostasis of the intermediate compartment and Golgi complex during cell stress.
Full-text · Article · Feb 2006 · The International Journal of Biochemistry & Cell Biology
[Show abstract][Hide abstract] ABSTRACT: In the present study we investigated whether apoptosis and phagocytosis are regulated by nuclear factor (NF)-kappaB in a model of chronic inflammation. The subcutaneous implant of lambda-carrageenin-soaked sponges elicited an inflammatory response, characterized by a time-related increase of leukocyte infiltration into the sponge and tissue formation, which was inhibited by simultaneous injection of wild-type oligodeoxynucleotide decoy to NF-kappaB. Molecular and morphological analysis performed on infiltrated cells demonstrated: 1) an inhibition of NF-kappaB/DNA binding activity; 2) an increase of polymorphonuclear leukocyte apoptosis correlated either to an increase of p53 or Bax and decrease of Bcl-2 protein expression; and 3) an increase of phagocytosis of apoptotic polymorphonuclear leukocytes by macrophages associated with an increase of transforming growth factor-beta1 and decrease of tumor necrosis factor-alpha as well as nitrite/nitrate production. Our results, showing that blockade of NF-kappaB by oligodeoxynucleotide decoy increases inflammatory cell apoptosis and phagocytosis, may contribute to lead to new insights into the mechanisms governing the inflammatory process.
Preview · Article · Aug 2004 · American Journal Of Pathology
[Show abstract][Hide abstract] ABSTRACT: To investigate protease activated receptor 2 (PAR-2) expression in human coronary atherosclerotic lesions because PAR-2 is involved in the modulation of inflammatory events and vascular function.
An immunohistochemical analysis was performed on serial arterial sections, using the following antibodies: MDA2, a murine monoclonal antibody against malondialdehyde lysine epitopes of oxidised low density lipoprotein (oxLDL); HAM-56, a monoclonal antibody against human macrophages/foam cells; B5, a rabbit polyclonal antibody against PAR-2; and SAM11, a mouse monoclonal antibody against human PAR-2. Sections containing at least one lesion showing substantial immunostaining were counted as positive, and results were expressed as per cent of all sections of the same artery.
PAR-2 expression was enhanced in human coronary atherosclerotic lesions. This phenomenon correlated with an increase in oxLDL epitopes in the coronary artery.
This study shows for the first time that PAR-2 expression is enhanced in human coronary atherosclerotic lesions, and suggests that PAR-2 dependent cellular trafficking may be one of the regulatory signalling responses to vascular injury. Further pharmacological studies will establish whether modulation (and in which direction) of PAR-2 represents a possible therapeutic target for controlling the vascular response to injury.
Full-text · Article · Jun 2004 · Journal of Clinical Pathology
[Show abstract][Hide abstract] ABSTRACT: Apoptosis of arterial cells induced by oxidized low-density lipoprotein (oxLDL) is thought to contribute to the progression of vascular dysfunction and atherogenesis. It is well established that diabetes mellitus is accompanied by both glycosylation and oxidation of LDL (glc-oxLDL), but the biological effects of these modified lipoproteins are poorly understood. We demonstrate here for the first time that glc-oxLDL increases TUNEL positivity and caspase-3 activation (by Western blot and immunocytochemistry) of human coronary smooth muscle cells. Overall, these effects induced by glc-oxLDL were greater than those achieved with oxLDL. Thus, glc-oxLDL activated downstream apoptotic signaling. This may influence the evolution of atherogenesis and vascular complications in diabetes.
No preview · Article · Jan 2004 · Annals of the New York Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Advanced stages of both cancer and atherosclerosis are characterized by a local increase in tissue mass that may be hard to control. This increase in tissue mass can be attributed to oxidation-sensitive modification of cell cycle-related events, including cellular proliferation, differentiation, and apoptosis, which could be secondary to alteration in the activity of tumor suppressor gene and oncogene products. The oncogene c-Myc has classically been considered to be involved in carcinogenesis and has more recently been implicated in both endothelial dysfunction and atherogenesis as well. Consequently, inhibition of c-Myc-dependent signaling has become a novel therapeutic opportunity and challenge in atherosclerosis and other cardiovascular diseases. Antioxidant strategies, RNA synthesis inhibitors such as mithramycin, and gene therapeutic approaches with antisense oligonucleotides against c-Myc are some of the promising strategies. In general, the increased biologic understanding of the participation of cell cycle events and targeting these events may enable to attenuate or prevent some of the complications of vascular and neoplastic diseases.
[Show abstract][Hide abstract] ABSTRACT: The massive increase in information on the human DNA sequence and the development of new technologies will have a profound impact on the diagnosis and treatment of cardiovascular diseases. The microarray is a micro-hybridisation based assay. The filter, called microchip or chip, is a special kind of membrane in which are spotted several thousands of oligonucleotides of cDNA fragments coding for known genes or expressed sequence tags. The resulting hybridisation signal on the chip is analysed by a fluorescent scanner and processed with a software package utilising the information on the oligonucleotide or cDNA map of the chip to generate a list of relative gene expression. Microarray technology can be used for many different purposes, most prominently to measure differential gene expression, variations in gene sequence (by analysing the genome of mutant phenotypes), or more recently, the entire binding site for transcription factors. Measurements of gene expression have the advantage of providing all available sequence information for any given experimental design and data interpretation in pursuit of biological understanding. This research tool will contribute to radically changing our understanding of cardiovascular diseases.
[Show abstract][Hide abstract] ABSTRACT: The beta(3)-adrenoreceptor plays a major role in lipolysis but the role and distribution of beta(3)-receptors in other specific sites have not been extensively studied. beta(3)-adrenergic receptors are present not only in adipose tissue but also in human gall bladder, colon, prostate, and skeletal muscle. Recently, beta(3)-adrenergic receptor stimulation was shown to elicit vasorelaxation of rat aorta through the NO-cGMP signal transduction pathway. Here we show that beta(3)-receptors are present in human corpus cavernosum and are localized mainly in smooth muscle cells. After activation by a selective beta(3)-adrenergic receptor agonist, BRL 37344, there was a cGMP-dependent but NO-independent vasorelaxation that was selectively blocked by a specific beta(3)-receptor antagonist. In addition, we report that the human corpus cavernosum exhibits basal beta(3)-receptor-mediated vasorelaxant tone and that beta(3)-receptor activity is linked to inhibition of the RhoA/Rho-kinase pathway. These observations indicate that beta(3)-receptors may play a physiological role in mediating penile erection and, therefore, could represent a therapeutic target for treatment of erectile dysfunction.
Full-text · Article · May 2003 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Peyronie's disease is the most frequent cause of penile curvature and occurs particularly in middle-age patients. The best technique for penile biopsy, in the evaluation of albuginea and cavernous tissue, has not been delineated yet. We present a new technique of penile biopsy, useful in the study of Peyronie's plaque, fibrosis and erectile dysfunction or any other pathological condition of the penis requiring a biopsy.
A treatment group (A) of 380 patients underwent Extra Shock Waves Treatment (ESWT) three times a week for 20 minutes, followed by a complete cycle of 12 injections of verapamil (10mg), every two weeks for six months. A control group (B) of 92 patients underwent verapamil injections alone. Three months after the end of the treatment, each patient underwent penile biopsy performed with Acu-Punch (Acuderm Inc.), a biopsy-punch armed with a well-sharpened rotating cylindrical blade, first used by dermatologists for cutaneous lesions.
A reduction of the plaque volume was found in 260/380 patients (68.4%) of group A and in 28/92 (30.4%) of group B; painful erection weaned off in 312/340 patients of group A (91.7%) and in 36/82 patients (43.9%) of group B. In all 472 patients an excellent specimen was obtained and both the tunica albuginea and the cavernous tissue were easily identified. In the 260 cases, in which the Extra Shock Waves Treatment was successful, scanning and transmission electron microscopy demonstrated a reduction in packing and clumping of the collagen fibers.
This new technique of penile biopsy with Acu-Punch can replace surgical biopsies when a surgical operation is not indicated. Such a low-invasive technique could be performed in all cases of Peyronie's disease and allows a more extensive use of microscopic analysis in the study of Peyronie's disease.
No preview · Article · Oct 2002 · European Urology
[Show abstract][Hide abstract] ABSTRACT: We investigated the role of constitutive transcription factor nuclear factor kappaB (NF-kappaB) in nitric oxide (NO)-mediated apoptosis in J774 macrophages. Our results show that NF-kappaB is present in untreated J774 cells in a form constitutively active. Incubation of cells with sodium nitroprusside (SNP) and S-nitroso-glutathione (GSNO), two NO-generating compounds, caused: (a) inhibition of constitutive NF-kappaB/DNA binding activity; (b) decrease of cell viability; (c) DNA fragmentation; (d) ApopTag positivity. Pyrrolidine dithiocarbamate (PDTC) and N-alpha-para-tosyl-L-lysine chloromethyl ketone (TLCK), two inhibitors of NF-kappaB activation, showed the same effects of both NO-generating compounds. Furthermore, SNP and GSNO as well as PDTC and TLCK significantly increased the cytoplasmic level of IkappaBalpha. All together these results demonstrate that constitutive NF-kappaB protects J774 macrophages from NO-induced apoptosis. Moreover, these findings show, for the first time, that NO-generating compounds may induce apoptosis in J774 macrophages by down-regulating constitutive NF-kappaB/DNA binding activity and suggest a novel mechanism by which NO induces apoptosis.
Full-text · Article · Mar 2001 · Cell Death and Differentiation
[Show abstract][Hide abstract] ABSTRACT: The main problem of using human corpus cavernosum (HCC) tissue to perform bioassay is linked to its limited availability further complicated by the heterogeneous source of the tissues used. Here, we show that gender reassignment is a reliable source of human tissue without major ethical problems. Indeed, the entire corpus cavernosum is obtained from the surgery procedure, which allows creating a standardized procedure to prepare HCC strip. In addition, human tissue, if kept in the fridge in the condition described, does not loose its ability to contract to phenylephrine (PE; alpha agonist), angiotensin II (AG II) and KCl up to 4 days. Furthermore, once contracted with PE, HCC relaxes to acetylcholine (endothelium-dependent mechanism); sodium nitroprusside (endothelium-independent mechanism); cromakalim (CRK), a K(ATP) channel opener; or alprostadil, a synthetic PGE2 (ALPR). In conclusion, we have standardized a procedure that allows the use of HCC strips to evaluate drug activity and/or to study pathophysiological mechanisms with an intact functional human tissue up to 4 days from the surgery procedure.
Full-text · Article · Nov 2000 · Journal of Pharmacological and Toxicological Methods