Amalia Forte

Upregulated TRPC1 Channel in Vascular Injury In Vivo and Its Role in Human Neointimal Hyperplasia

Occlusive vascular disease is a widespread abnormality leading to lethal or debilitating outcomes such as myocardial infarction and stroke. It is part of atherosclerosis and is evoked by clinical procedures including angioplasty and grafting of saphenous vein in bypass surgery. A causative factor is the switch in smooth muscle cells to an invasive and proliferative mode, leading to neointimal hyperplasia. Here we reveal the importance to this process of TRPC1, a homolog of Drosophila transient receptor potential. Using 2 different in vivo models of vascular injury in rodents we show hyperplasic smooth muscle cells have upregulated TRPC1 associated with enhanced calcium entry and cell cycle activity. Neointimal smooth muscle cells after balloon angioplasty of pig coronary artery also express TRPC1. Furthermore, human vein samples obtained during coronary artery bypass graft surgery commonly exhibit an intimal structure containing smooth muscle cells that expressed more TRPC1 than the medial layer cells. Veins were organ cultured to allow growth of neointimal smooth muscle cells over a 2-week period. To explore the functional relevance of TRPC1, we used a specific E3-targeted antibody to TRPC1 and chemical blocker 2-aminoethoxydiphenyl borate. Both agents significantly reduced neointimal growth in human vein, as well as calcium entry and proliferation of smooth muscle cells in culture. The data suggest upregulated TRPC1 is a general feature of smooth muscle cells in occlusive vascular disease and that TRPC1 inhibitors have potential as protective agents against human vascular failure.

ROLE OF MYOFIBROBLASTS IN VASCULAR REMODELLING: FOCUS ON RESTENOSIS AND ANEURYSM

Myofibroblasts (MFs) are contractile cells deriving from a multiplicity of resident cells and/or circulating progenitors that are known to play a key role in wound healing. They were first discovered and analysed in the early 1970s in granulation tissue. Since their first identification, the role of MF and their mechanisms of differentiation have been highlighted in a number of diseases, including organ fibrosis and tumours, with particular attention devoted to the liver, kidney, and pulmonary fibrosis. The aim of this review is to summarize the current evidence for the role played by MFs in two frequent vascular diseases related to the remodelling of the vascular wall: the different forms of arterial restenosis and the most common forms of thoracic aortic aneurysm. The in-depth knowledge of the molecular pathways involved in MF differentiation, contraction, and survival/apoptosis could contribute to the identification of novel therapeutic strategies for anti-fibrotic and anti-remodelling therapy of vascular diseases in which these cells are involved.

Expression Pattern of Stemness-Related Genes in Human Endometrial and Endometriotic Tissues

Endometriosis is a chronic disease characterized by the presence of ectopic endometrial tissue outside of the uterus with mixed traits of benign and malignant pathology. In this study we analyzed in endometrial and endometriotic tissues the differential expression of a panel of genes that are involved in preservation of stemness status and consequently considered as markers of stem cell presence. The expression profiles of a panel of 13 genes (SOX2, SOX15, ERAS, SALL4, OCT4, NANOG, UTF1, DPPA2, BMI1, GDF3, ZFP42, KLF4, TCL1) were analyzed by reverse transcription-polymerase chain reaction in human endometriotic (n = 12) and endometrial samples (n = 14). The expression of SALL4 and OCT4 was further analyzed by immunohistochemical methods. Genes UTF1, TCL1, and ZFP42 showed a trend for higher frequency of expression in endometriosis than in endometrium (P< 0.05 for UTF1), whereas GDF3 showed a higher frequency of expression in endometrial samples. Immunohistochemical analysis revealed that SALL4 was expressed in endometriotic samples but not in endometrium samples, despite the expression of the corresponding mRNA in both the sample groups. This study highlights a differential expression of stemness-related genes in ectopic and eutopic endometrium and suggests a possible role of SALL4-positive cells in the pathogenesis of endometriosis.

Small Interfering RNAs and Antisense Oligonucleotides for Treatment of Neurological Diseases

The complexity of the central nervous system (CNS) exposes it to a number of different diseases, often caused by only small variations in gene sequence or expression level. Antisense oligonucleotides and RNA interference-mediated therapies hold great promise for the treatment of CNS diseases in which neurodegeneration is linked to overproduction of endogenous protein or to synthesis of aberrant proteins coded by dominant mutant alleles. Nevertheless, difficulties related to the crossing of the blood-brain barrier, expression vectors, molecule design and to the choosing of the correct target, should be effectively solved. This review summarizes some of the most recent findings concerning the administration of potential nucleic acid-based therapeutic drugs, as well as the most promising studies performed both in vitro and in animal models of disease. Finally, some current clinical trials involving antisense oligonucleotides or silencing RNA for therapy of neurological disorders are illustrated. Results of current studies and clinical trials are exciting, and further results will be certainly reached with increasing knowledge of blood-brain barrier transporters, of genes involved in neurological disease and in new vectors for efficient delivery to brain.

Mesenchymal stem cells effectively reduce surgically induced stenosis in rat carotids

Restenosis following vascular injury remains a pressing clinical problem. Mesenchymal stem cells (MSCs) promise as a main actor of cell‐based therapeutic strategies. The possible therapeutic role of MSCs in vascular stenosis in vivo has been poorly investigated so far. We tested the effectiveness of allogenic bone marrow‐derived MSCs in reduction of stenosis in a model of rat carotid arteriotomy. MSCs were expanded in vitro retaining their proliferative and differentiation potentiality. MSCs were able to differentiate into adipocyte and osteocyte mesenchymal lineage cells, retained specific antigens CD73, CD90, and CD105, expressed smooth muscle alpha‐actin, were mainly in proliferative phase of cell cycle and showed limited senescence. WKY rats were submitted to carotid arteriotomy and to venous administration with 5 × 106 MSCs. MSCs in vivo homed in injured carotids since 3 days after arteriotomy but not in contralateral uninjured carotids. Lumen area in MSC‐treated carotids was 36% greater than in control arteries (P = 0.016) and inward remodeling was limited in MSC‐treated carotids (P = 0.030) 30 days after arteriotomy. MSC treatment affected the expression level of inflammation‐related genes, inducing a decrease of IL‐1β and Mcp‐1 and an increase of TGF‐β in injured carotids at 3 and 7 days after arteriotomy (P < 0.05). Taken together, these results indicate that allogenic MSC administration limits stenosis in injured rat carotids and plays a local immunomodulatory action. J. Cell. Physiol. 217: 789–799, 2008.

Molecular analysis of arterial stenosis in rat carotids

A new model of surgical injury for the induction and development of stenosis in common rat carotids is described. This model differs from balloon angioplasty or vein graft systems currently applied on animals to develop stenosis, since it involves the entire vessel wall layers and mimics the injury occurring during arterial grafts, endarterectomy or organ transplantation. At different times following arterial damage, the pattern of expression of genes already known to be involved in the proliferation, differentiation, and apoptosis of smooth muscle cells (c‐myc, Angiotensin II receptor 1, Bcl‐2 and Bax α), as well as of Rb and Rb2 genes, whose pattern of expression after arterial injury has not yet been reported, was analyzed by semi‐quantitative reverse transcription‐polymerase chain reaction technique. Histological and histochemical analysis on carotid sections shows the morphological changes which occurred 30 days after surgical injury in the vessel wall. Molecular and histological data demonstrate that this model of surgical injury induces neointimal proliferation in about 30% of rats. In about 70% of the remaining rats, it induces the processes responsible for negative remodelling, namely the significant accumulation of extracellular matrix and fibers and disorganization of arterial tunics. This model is therefore available for further studies on the expression of genes involved in the arterial stenotic process, as well as for testing drugs aimed at limiting this recurrent pathophysiological phenomenon. J. Cell. Physiol. 186:307–313, 2001.

Genetic, epigenetic and stem cell alterations in endometriosis: new insights and potential therapeutic perspectives.

Human endometrium is a highly dynamic tissue, undergoing periodic growth and regression at each menstrual cycle. Endometriosis is a frequent chronic pathological status characterized by endometrial tissue with an ectopic localization, causing pelvic pain and infertility and a variable clinical presentation. In addition, there is well-established evidence that, although endometriosis is considered benign, it is associated with an increased risk of malignant transformation in approximately 1.0% of affected women, with the involvement of multiple pathways of development. Increasing evidence supports a key contribution of different stem/progenitor cell populations not only in the cyclic regeneration of eutopic endometrium, but also in the pathogenesis of at least some types of endometriosis. Evidence has arisen from experiments in animal models of disease through different kinds of assays (including clonogenicity, the label-retaining cell approach, the analysis of undifferentiation markers), as well as from descriptive studies on ectopic and eutopic tissue samples harvested from affected women. Changes in stem cell populations in endometriotic lesions are associated with genetic and epigenetic alterations, including imbalance of miRNA expression, histone and DNA modifications and chromosomal aberrations. The present short review mainly summarizes the latest observations contributing to the current knowledge regarding the presence and the potential contribution of stem/progenitor cells in eutopic endometrium and the aetiology of endometriosis, together with a report of the most recently identified genetic and epigenetic alterations in endometriosis. We also describe the potential advantages of single cell molecular profiling in endometrium and in endometriotic lesions. All these data can have clinical implications and provide a basis for new potential therapeutic applications.

c-Myc Antisense Oligonucleotides Preserve Smooth Muscle Differentiation and Reduce Negative Remodelling following Rat Carotid Arteriotomy

Objectives: The vascular biology of restenosis is complex and not fully understood, thus explaining the lack of effective therapy for its prevention in clinical settings. The role of c-Myc in arteriotomy-induced stenosis, smooth muscle cell (SMC) differentiation and apoptosis was investigated in rat carotids applying full phosphorothioate antisense (AS) oligonucleotides (ODNs). Methods: Carotid arteries from WKY rats were submitted to arteriotomy and to local application of ODNs through pluronic gel. Apoptosis (deoxynucleotidyl transferase-mediated dUTP nick end-labelling), SMC differentiation (SM22 immunofluorescence) and vessel morphology and morphometry (image analysis) were determined 2, 5 and 30 days after injury, respectively. Results: AS ODNs induced a 60% decrease of target c-Myc mRNA 4 h after surgery in comparison to control sense (S) and scrambled ODN-treated carotids (p < 0.05). A significant 37 and 50% decrease in SM22 protein in the media of S ODN-treated and untreated carotids was detected when compared to uninjured contralateral arteries (p < 0.05). This reduction in SM22 expression was prevented in AS ODN-treated carotids. Stenosis was mainly due to adventitial constrictive remodelling. Lumen area in AS ODN-treated carotids was 35% greater than in control arteries 30 days after surgery (p < 0.05). TUNEL assay revealed increased apoptosis in AS ODN-treated carotids (p < 0.05). Conclusions: c-Myc AS ODNs reduce arteriotomy-induced negative remodelling. This is accompanied by maintained SMC differentiation and greater apoptosis. The combination of reduced c-Myc-induced proliferation and increased apoptosis may thus underlie the less severe remodelling upon treatment with c-Myc mRNA AS ODN.

Novel potential targets for prevention of arterial restenosis: insights from the pre-clinical research.

Restenosis is the pathophysiological process occurring in 10-15% of patients submitted to revascularization procedures of coronary, carotid and peripheral arteries. It can be considered as an excessive healing reaction of the vascular wall subjected to arterial/venous bypass graft interposition, endarterectomy or angioplasty. The advent of bare metal stents, drug-eluting stents and of the more recent drug-eluting balloons, have significantly reduced, but not eliminated, the incidence of restenosis, which remains a clinically relevant problem. Biomedical research in pre-clinical animal models of (re)stenosis, despite its limitations, has contributed enormously to the identification of processes involved in restenosis progression, going well beyond the initial dogma of a primarily proliferative disease. Although the main molecular and cellular mechanisms underlying restenosis have been well described, new signalling molecules and cell types controlling the progress of restenosis are continuously being discovered. In particular, microRNAs and vascular progenitor cells have recently been shown to play a key role in this pathophysiological process. In addition, the advanced highly sensitive high-throughput analyses of molecular alterations at the transcriptome, proteome and metabolome levels occurring in injured vessels in animal models of disease and in human specimens serve as a basis to identify novel potential therapeutic targets for restenosis. Molecular analyses are also contributing to the identification of reliable circulating biomarkers predictive of post-interventional restenosis in patients, which could be potentially helpful in the establishment of an early diagnosis and therapy. The present review summarizes the most recent and promising therapeutic strategies identified in experimental models of (re)stenosis and potentially translatable to patients subjected to revascularization procedures.

Expression profiles in surgically-induced carotid stenosis: a combined transcriptomic and proteomic investigation.

Vascular injury aimed at stenosis removal induces local reactions often leading to restenosis. The aim of this study was a concerted transcriptomic‐proteomics analysis of molecular variations in a model of rat carotid arteriotomy, to dissect the molecular pathways triggered by vascular surgical injury and to identify new potential anti‐restenosis targets. RNA and proteins extracted from inbred Wistar Kyoro (WKY) rat carotids harvested 4 hrs, 48 hrs and 7 days after arteriotomy were analysed by Affymetrix rat microarrays and by bidi‐mensional electrophoresis followed by liquid chromatography and tandem mass spectrometry, using as reference the RNA and the proteins extracted from uninjured rat carotids. Results were classified according to their biological function, and the most significant Kyoro Encyclopedia of Genes and Genomes (KEGG) pathways were identified. A total of 1163 mRNAs were differentially regulated in arteriotomy‐injured carotids 4 hrs, 48 hrs and 7 days after injury (P < 0.0001, fold‐change ≥2), while 48 spots exhibited significant changes after carotid arteriotomy (P < 0.05, fold‐change ≥2). Among them, 16 spots were successfully identified and resulted to correspond to a set of 19 proteins. mRNAs were mainly involved in signal transduction, oxidative stress/inflammation and remodelling, including many new potential targets for limitation of surgically induced (re)stenosis (e.g. Arginase I, Kruppel like factors). Proteome analysis confirmed and extended the microrarray data, revealing time‐dependent post‐translational modifications of Hsp27, haptoglobin and contrapsin‐like protease inhibitor 6, and the differential expression of proteins mainly involved in contractility. Transcriptomic and proteomic methods revealed functional categories with different preferences, related to the experimental sensitivity and to mechanisms of regulation. The comparative analysis revealed correlation between transcriptional and translational expression for 47% of identified proteins. Exceptions from this correlation confirm the complementarities of these approaches.