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Adaptive immunity is typified by specificity and memory. Immune memory protects from subsequent infection and relies on functional, fully activated, T lymphocytes. Mesenchymal stromal cells (MSC) have been investigated for their potential therapeutic applications in a variety of diseases in which a dysregulated immune response plays a central role in pathogenesis or progression. Notably, for adaptive immunity, MSC can reduce the activation and cytotoxic activity of CD8+T lymphocytes as well as the polarization of CD4+T lymphocytes toward inflammatory subsets while favoring polarization toward the T regulatory subset. Although MSC have been widely reported to impact CD4 T cell proliferation and polarization, how MSC affect T-cell commitment toward memory subsets is still not known. Here, we report for the first time that MSC isolated from the amniotic membrane of human term placenta (hAMSC) determine T cell fate. We show that hAMSC influence naïve CD8+T cell activation and differentiation by downregulating mTOR pathway activation and modulating the expression of Tbet and Eomes, master regulators of the commitment of naïve CD8+T cells toward memory precursor effector cells (MPECs). This effect can be partly attribute to the ability of hAMSC to reduce the phosphorylation of STAT4 and STAT5, two transcriptional factors downstream IL-12Rβ1 and IL-2Rα receptors. Our results unravel a novel feature of MSC, offering new mechanistic insights into the effects of MSC in the treatment of diseases characterized by an altered activation of memory subsets, such as autoimmune diseases and graft versus host disease.

Background Duchenne muscular dystrophy (DMD) is a muscle disease caused by mutations in the dystrophin gene characterized by myofiber fragility and progressive muscle degeneration. The genetic defect results in a reduced number of self-renewing satellite cells (MuSCs) and an impairment of their activation and differentiation which lead to the exhaustion of skeletal muscle regeneration potential and muscle replacement by fibrotic and fatty tissue. In this study we focused on an unexplored strategy to improve MuSC function and their niche based on the regenerative properties of mesenchymal stromal cells from the amniotic membrane (hAMSCs), multipotent cells that have been recognized to have a role in tissue repair in different disease models. Methods The myogenic capacity of the hAMSC secretome (CM-hAMSC) and of their extracellular vesicles (EVs) were tested on human myoblasts, muscle fibers and dystrophic muscle stem cells in terms of ability to favor MuSC activation and differentiation both in vitro and in vivo. Specifically, the regenerative ability of EV-hAMSC was assessed by histological and molecular analyses after intra-muscular injection in the mouse model of DMD –mdx-. Results We demonstrate that CM-hAMSC and EVs isolated thereof are able to directly stimulate the in vitro proliferation and differentiation of human myoblasts and mouse MuSC from dystrophic muscles. Indeed, they significantly increased both nuclei numbers (pConclusions In this study, for the first time, we demonstrate that the hAMSC secretome (CM-hAMSC) and mainly EVs derived thereof are able to promote the muscle regeneration potential supporting proliferation and differentiation of resident muscle stem cells. This study paves the way to develop a novel regenerative treatment to counteract DMD progression based on the capacity of EV-hAMSC to reduce fibrosis and enhance myogenesis in dystrophic muscles.

The main objective of this book is to present a thorough update on stem cell research and the potential therapeutic applications of stem cells. The text is structured following a path that starts from the molecular basics and the biological properties of pluripotent, embryonic or reprogrammed stem cells, and it compares the different degrees of stemness, while describing the adult stem populations residing in the various tissues and organs of the human body. Starting from basic research, the book discusses examples of regenerative medicine that translate the experimental findings into clinical applications of cell therapy. Finally, the book reviews how stem cells represent a model to understand not only the physiological mechanisms that control their fate, but also the pathological mechanisms involved in the aberrant biology of cancer stem cells. Each chapter has been conceived by distinguished researchers in the field who provide detailed and updated contributions that distill knowledge in a very readable text. Laura Bonsi is Associate Professor of Histology at the Department of Experimental, Diagnostic and Specialty Medicine of the University of Bologna. Francesco Alviano is Assistant Professor of Histology at the Department of Experimental, Diagnostic and Specialty Medicine of the University of Bologna. Prof. Bonsi and Dr. Alviano are authors of numerous scientific papers published in international journals. They coordinate the Biotechnology Laboratory of Human Stem Cells at the University of Bologna, that is focused on the study of stem cells isolated from different sources, above all from extraembryonic tissues such as the placenta, the umbilical cord, and the fetal membranes. They are longterm members of the International Placenta Stem Cell Society (IPLASS).

The pathophysiology of preeclampsia (PE) is poorly understood; however, there is a large body of evidence that suggests a role of immune cells in the development of PE. Amongst these, B cells are a dominant element in the pathogenesis of PE, and they have been shown to play an important role in various immune-mediated diseases, both as pro-inflammatory and regulatory cells. Perinatal cells are defined as cells from birth-associated tissues isolated from term placentas and fetal annexes and more specifically from the amniotic membrane, chorionic membrane, chorionic villi, umbilical cord (including Wharton’s jelly), the basal plate, and the amniotic fluid. They have drawn particular attention in recent years due to their ability to modulate several aspects of immunity, making them promising candidates for the prevention and treatment of various immune-mediated diseases. In this review we describe main findings regarding the multifaceted in vitro and in vivo immunomodulatory properties of perinatal cells, with a focus on B lymphocytes. Indeed, we discuss evidence on the ability of perinatal cells to inhibit B cell proliferation, impair B cell differentiation, and promote regulatory B cell formation. Therefore, the findings discussed herein unveil the possibility to modulate B cell activation and function by exploiting perinatal immunomodulatory properties, thus possibly representing a novel therapeutic strategy in PE.

Growing evidence suggests a mechanistic link between inflammation and the development and progression of fibrotic processes. Mesenchymal stromal cells derived from the human amniotic membrane (hAMSCs), which display marked immunomodulatory properties, have been shown to reduce bleomycin‐induced lung fibrosis in mice, possibly by creating a microenvironment able to limit the evolution of chronic inflammation to fibrosis. However, the ability of hAMSCs to modulate immune cells involved in bleomycin‐induced pulmonary inflammation has yet to be elucidated. Herein, we conducted a longitudinal study of the effects of hAMSCs on alveolar and lung immune cell populations upon bleomycin challenge. Immune cells collected through bronchoalveolar lavage were examined by flow cytometry, and lung tissues were used to study gene expression of markers associated with different immune cell types. We observed that hAMSCs increased lung expression of T regulatory cell marker Foxp3, increased macrophage polarization toward an anti‐inflammatory phenotype (M2), and reduced the antigen‐presentation potential of macrophages and dendritic cells. For the first time, we demonstrate that hAMSCs markedly reduce pulmonary B‐cell recruitment, retention, and maturation, and counteract the formation and expansion of intrapulmonary lymphoid aggregates. Thus, hAMSCs may hamper the self‐maintaining inflammatory condition promoted by B cells that continuously act as antigen presenting cells for proximal T lymphocytes in injured lungs. By modulating B‐cell response, hAMSCs may contribute to blunting of the chronicization of lung inflammatory processes with a consequent reduction of the progression of the fibrotic lesion., This study demonstrates that amniotic mesenchymal stromal cells reduce pulmonary fibrosis in bleomycin‐challenged mice by creating an anti‐inflammatory microenvironment mediated by their ability to control B‐cell recruitment, retention, maturation, and to reduce the formation/expansion of lymphoid aggregates in diseased lungs.

The tumor microenvironment (TME) plays a critical role in tumorigenesis and is composed of different cellular components, including immune cells and mesenchymal stromal cells (MSCs). In this review, we will discuss MSCs in the TME setting and more specifically their interactions with immune cells and how they can both inhibit (immunosurveillance) and favor (immunoediting) tumor growth. We will also discuss how MSCs are used as a therapeutic strategy in cancer. Due to their unique immunomodulatory properties, MSCs isolated from perinatal tissues are intensely explored as therapeutic interventions in various inflammatory-based disorders with promising results. However, their therapeutic applications in cancer remain for the most part controversial and, importantly, the interactions between administered perinatal MSC and immune cells in the TME remain to be clearly defined.

Placenta-derived mesenchymal stromal cells (MSC) have often been considered to linger behind their equivalents from other tissues, such as MSC from bone marrow, in many aspects including their therapeutic potential in regenerative medicine. Nowadays however, it is clear that certain aspects make placental MSC attractive as a cellular therapy, such as their lack of ethical concerns and ease of isolation from human term placenta, a material long regarded as biological waste. Moreover, placental MSC virtually lack expression of human leukocyte antigens and co-stimulatory molecules, making them very attractive for transplantation in allogeneic settings. In the context of cancer, cell therapy remains an area of intense investigation whereby MSC have been shown to play opposing roles, and placental MSC are no exception. In this review, we will discuss dichotomy of placental MSC that underscores the challenges in understanding their therapeutic potential in oncology.

During pregnancy, a successful coexistence between the mother and the semi-allogenic fetus occurs which requires a dynamic immune system to guarantee an efficient immune protection against possible infections and tolerance toward fetal antigens. The mechanism of fetal-maternal tolerance is still an open question. There is growing in vitro and in vivo evidence that mesenchymal stromal cells (MSC) which are present in perinatal tissues have a prominent role in generating a functional microenvironment critical to a successful pregnancy. This review highlights the immunomodulatory properties of perinatal MSC and their impact on the major immune cell subsets present in the uterus during pregnancy, such as natural killer cells, antigen-presenting cells (macrophages and dendritic cells), and T cells. Here, we discuss the current understanding and the possible contribution of perinatal MSC in the establishment of fetal-maternal tolerance, providing a new perspective on the physiology of gestation.

Macroautophagy/autophagy occurs at basal levels in all eukaryotic cells and plays an important role in maintaining bio-energetic homeostasis through the control of molecule degradation and organelle turnover. It can be induced by environmental conditions such as starvation, and is deregulated in many diseases including autoimmune diseases, neurodegenerative disorders, and cancer. Interestingly, the modulation of autophagy in mesenchymal stem cells (MSCs) represents a possible mechanism which, affecting MSC properties, may have an impact on their regenerative, therapeutic potential. Furthermore, the ability of MSCs to modulate autophagy of cells in injured tissues/organs has been recently proposed to be involved in the regeneration of damaged tissues and organs. In particular, MSCs can affect autophagy in immune cells involved in injury-induced inflammation reducing their survival, proliferation, and function and favoring the resolution of inflammation. In addition, MSCs can affect autophagy in endogenous adult or progenitor cells, promoting their survival, proliferation and differentiation supporting the restoration of functional tissue. This review provides, for the first time, an overview of the studies which highlight a possible link between the therapeutic properties of MSCs and their ability to modulate autophagy, and it summarizes examples of disorders where these therapeutic properties have been correlated with such modulation. A better elucidation of the mechanism(s) through which MSCs can modulate the autophagy of target cells and how autophagy can affect MSCs therapeutic properties, can provide a wider perspective for the clinical application of MSCs in the treatment of many diseases.Abbreviations: 3-MA: 3-methyladenine; AD: Alzheimer disease; ATG: autophagy-related; BECN1: beclin 1; BM: bone marrow; CD: cluster of differentiation; EAE: experimental autoimmune encephalomyelitis; IL: interleukin; INF: interferon; LAP: LC3-associated phagocytosis; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MSCs: mesenchymal stem cells; MTOR: mechanistic target of rapamycin kinase; PD: Parkinson disease; PtdIns3K: class III phosphatidylinositol 3-kinase; ROS: reactive oxygen species; SLE: systemic lupus erythematosus; SQSTM1: sequestosome 1; TBI: traumatic brain injury; TGF: transforming growth factor; TNF: tumor necrosis factor.

Liver fibrosis is characterized by excessive accumulation of extracellular matrix components in the liver parenchyma that distorts the normal architecture and hepatic function. Progressive fibrosis could end in the advanced stage known as cirrhosis, resulting in the need to resort to liver transplantation. Amniotic membrane (AM) has emerged as an innovative therapeutic approach for chronic liver diseases due to its anti-inflammatory, antiscarring, and wound-healing effects. We have recently shown that AM can be used as a patch on the liver surface at the same time of fibrosis induction, resulting in significantly reduced progression and severity of biliary fibrosis. Here we investigated the effects of human AM on the established rat model of liver fibrosis, induced by the bile duct ligation (BDL). We also explored the effect of AM on the expression of transforming growth factor-β1 (TGF-β1), the main profibrogenic factor in hepatic fibrosis, and the proinflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and anti-inflammatory cytokine IL-10. Two weeks after BDL, the liver was covered with a fragment of AM or left untreated. Six weeks later, the fibrosis was first assessed by the semiquantitative Knodell and the METAVIR scoring systems and, thereafter, by CellProfiler digital image analysis to quantify the area occupied by collagen deposition, ductular reactions (DRs), activated myofibroblasts, and TGF-β1. The hepatic cytokines were determined by ELISA. AM-treated rats showed a significantly lower score compared to the control BDL rats (2.5 ± 0.9 vs. 3.5 ± 0.3, respectively; p < 0.05). The collagen deposition, DRs, number of activated myofibroblasts, and TGF-β1 were all reduced to about 50% of levels observed in untreated BDL rats. These findings suggest that AM, when applied as a patch onto the liver surface, is useful for treating well-established cholestatic fibrosis, and the mechanism was partly by means of downregulating the profibrotic factor TGF-β1 and IL-6.

Different in vitro and in vivo studies have demonstrated that cells isolated from the amniotic membrane possess broad immunomodulatory activities. Indeed, both amniotic epithelial cells (hAECs) and amniotic mesenchymal stromal cells (hAMSCs) suppress B cell and T cell proliferation, generation of Th1, Th17, and cytotoxic T cells, and production of inflammatory cytokines. Moreover, both hAMSCs and hAECs suppress inflammatory activities of monocytes, macrophages, dendritic cells, neutrophils, and natural killer cells, while promoting the induction of cells with regulatory functions such as regulatory T cells and antiinflammatory M2 macrophages. Increasing evidence shows that these immunomodulatory properties are the mechanism through which amniotic cells can exert the therapeutic effect observed in the preclinical setting of diseases characterized by an exacerbated inflammatory response. In this chapter, we will comprehensively review in vitro and in vivo studies of the immunomodulatory properties of hAECs and hAMSCs and discuss differences between the two cell populations.

Myofibroblasts are key fibrogenic cells responsible for excessive extracellular matrix synthesis characterizing the fibrotic lesion. In liver fibrosis, myofibroblasts derive either from activation of hepatic stellate cells (HSC) and portal fibroblasts (PF), or from the activation of fibroblasts that originate from ductular epithelial cells undergoing epithelial–mesenchymal transition. Ductular cells can also indirectly promote myofibroblast generation by activating TGF‐β, the main fibrogenic growth factor, through αvβ6 integrin. In addition, after liver injury, liver sinusoidal cells can lose their ability to maintain HSC quiescence, thus favouring HSC differentiation towards myofibroblasts. The amniotic membrane and epithelial cells (hAEC) derived thereof have been shown to decrease hepatic myofibroblast levels in rodents with liver fibrosis. In this study, in a rat model of liver fibrosis, we investigated the effects of hAEC on resident hepatic cells contributing to myofibroblast generation. Our data show that hAEC reduce myofibroblast numbers with a consequent reduction in fibronectin and collagen deposition. Interestingly, we show that hAEC strongly act on specific myofibroblast precursors. Specifically, hAEC reduce the activation of PF rather than HSC. In addition, hAEC target reactive ductular cells by inhibiting their proliferation and αvβ6 integrin expression, with a consequent decrease in TGF‐β activation. Moreover, hAEC counteract the transition of ductular cells towards fibroblasts, while it does not affect injury‐induced and fibrosis‐promoting sinusoidal alterations. In conclusion, among the emerging therapeutic applications of hAEC in liver diseases, their specific action on PF and ductular cells strongly suggests their application in liver injuries involving the expansion and activation of the portal compartment.

Regenerative medicine aims to repair and regenerate damaged cells, tissues, and organs in order to restore function. Regeneration can be obtained either by cell replacement or by stimulating the body's own repair mechanisms. Importantly, a favorable environment is required before any regenerative signal can stimulate resident stem/stromal cells, and regeneration is possible only after the resolution of injury-induced inflammation. An exacerbated immune response is often present in cases of degenerative, inflammatory-based diseases. Here we discuss how amniotic membrane cells, and their derivatives, can contribute to the resolution of many diseases with altered immune response by acting on different inflammatory mediators.

Background and aims. We have demonstrated recently that transplantation of placental membrane-derived cells reduces bleomycin-induced lung fi brosis in mice, despite a limited presence of transplanted cells in host lungs. Because placentaderived cells are known to release factors with potential immunomodulatory and trophic activities, we hypothesized that transplanted cells may promote lung tissue repair via paracrine-acting molecules. To test this hypothesis, we examined whether administration of conditioned medium (CM) generated from human amniotic mesenchymal tissue cells (AMTC) was able to reduce lung fi brosis in this same animal model. Methods. Bleomycin-challenged mice were either treated with AMTC-CM or control medium, or were left untreated (Bleo group). After 9 and 14 days, the distribution and severity of lung fi brosis were assessed histologically with a scoring system. Collagen deposition was also evaluated by quantitative image analysis. Results. At day 14, lung fi brosis scores in AMTC-CM-treated mice were signifi cantly lower ( P � 0.05) compared with mice of the Bleo group, in terms of fidistribution [1.0 (interquartile range, IQR 0.9) versus 3.0 (IQR 1.8)], fi broblast proliferation [0.8 (IQR 0.4) versus 1.6 (IQR 1.0)], collagen deposition [1.4 (IQR 0.5) versus 2.0 (IQR 1.2)] and alveolar obliteration [2.3 (IQR 0.8) versus 3.2 (IQR 0.5)]. No differences were observed between mice of the Bleo group and mice treated with control medium. Quantitative analysis of collagen deposition confi rmed these fi ndings. Importantly, AMTC-CM treatment signifi cantly reduced the fi brosis progression between the two observation time-points. Conclusions. This pilot study supports the notion that AMTC exert anti-fi brotic effects through release of yet unknown soluble factors.

Intracoronary infusion of bone marrow cells (BMCs) is thought to induce cardiac regeneration in ischemic heart disease and dilated cardiomyopathy. The aim of our study was to develop a new method to inject BMCs into coronary arteries of small experimental animals. Transient atrioventricular block (AVB) was induced in 25 rats and 39 hamsters by intracarotid injection of adenosine 5′-triphosphate (ATP). Contrast echocardiography was obtained. BMCs (0.2–0.5 ml) were collected through femoral puncture, stained with PKH26 and injected into the carotid artery (CA). Animals were immediately sacrificed or followed for 1 month. To evaluate BMCs transfer from CA to myocardium, AVB and BMCs injections were performed in 10 hamsters subjected to coronary ligation for 30 min. Induction of transient AVB was possible in all animals by injecting 20–30 mg of ATP. Animals recovered a basal cardiac activity spontaneously or by dopamine injection. Flash injection of contrast medium through the CA induced staining of aortic root, coronary arteries, and myocardium. BMCs injection was possible in all cases. No immediate or late ECG changes were observed. Immediately after injection in healthy animals, histological examination showed the presence of BMCs in small coronary arteries and, after 1 month, the absence of infarction. In ischemic hearts, the presence of BMCs in the myocardium was observed 24 h after ischemia. ATP-induced AVB block allows for percutaneous intracoronary injection of BMCs in small experimental animals with no immediate or late mortality and morbidity. This method offers new perspectives for the investigation of BMCs coronary infusion and engraftment in heart diseases.

In the 1800’s, a baby born with a caul, a remnant of the amniotic sack or fetal membranes, was thought to be lucky, special, or protected. Over time, fetal membranes lost their legendary power and were soon considered nothing more than biological waste after birth. However, placenta tissues have reclaimed their potential and since the early 1900’s an increasing body of evidence has shown that these tissues have clinical benefits in a wide range of wound repair and surgical applications. Nowadays, there is a concerted effort to understand the mechanisms underlying the beneficial effects of placental tissues, and, more recently, cells derived thereof. This review will summarize the historical and current clinical applications of human placental tissues, and cells isolated from these tissues, and discuss some mechanisms thought to be responsible for the therapeutic effects observed after tissue and/or cell transplantation.

New avenues in the general area of research into stem cells have been opened in recent years through the discovery that cells with stem properties can be isolated from the human placenta, which represents an ethically sound, easily procured and plentiful source, and is therefore an attractive alternative to other stem cell sources. In particular, the human amniotic membrane, which already has a long history of use as a surgical material, is also proving to be a valuable reserve of cells for research and development of novel therapeutic approaches for regenerative/reparative medicine. In this chapter we will describe the two main amniotic membrane-derived cell populations, i.e. epithelial cells and mesenchymal stromal/stem cells, and some of their sub-populations, in terms of their stem properties which have been discovered to date. We will explore reasons why these amniotic membrane-derived cell populations cannot be considered “true” stem cells, at least in the classical sense of the term, even though they have some features of progenitor-like cells and certainly display some very interesting biological properties both for research purposes and for potential clinical applications. We will also bring into discussion the fact that much still remains to be further investigated and even discovered in this evolving research field, although despite this, the very promising results achieved so far are certainly very encouraging as a basis for future studies.

Ivabradine, the first representative of a new class of exclusive heart rate-reducing agents, selectively inhibits the I(f) current in the sinoatrial node. The direct electrophysiological consequence of this inhibition is a reduction in the slope of the diastolic depolarisation curve and a decrease in heart rate. Pharmacological inhibition of the I(f) current with ivabradine has been shown to preserve coronary vasodilatation upon exercise, i.e., myocardial perfusion, with no negative inotropic effects and maintenance of cardiac contractility. Ivabradine protects the myocardium during ischaemia, improves left ventricular function in congestive heart failure, and reduces remodelling subsequent to myocardial infarction. Pure heart rate reduction by specific and selective I(f) inhibition decreases oxygen demand, improves myocardial energetics and improves perfusion of the ischaemic myocardium. We can expect distinct clinical benefits from long-term heart rate reduction in patients with chronic ischaemic disease.

Aim Immune activation in severe congestive heart failure (CHF) due to idiopathic conditions is a well-known phenomenon. The question remains: is it confined to a specific aetiology or is a disease-dependent derangement? We investigated immune alterations in ischaemic compared to idiopathic patients with CHF and healthy subjects. Methods and results We evaluated alterations of immune activation studying both in vivo and in vitro parameters. In both idiopathic and ischaemic CHF patients: (a) lymphocyte count was reduced (P < 0.01); (b) lymphocyte subsets were altered (CD4/CD8 ratio increased vs. controls: 2.53 ± 0.8 and 3.3 ± 2.1% in idiopathic and ischaemic patients, vs. 1.8 ± 0.6% in controls; P < 0.01); (c) lymphocytes from patients underwent a higher apoptosis (27.1 ± 4.3% in idiopathic CHF and 25.4 ± 3.5% in ischaemic CHFvs. 4.5 ± 1.4%; P < 0.0001); (d) TNF-a production from lymphocytes and monocytes of patients was higher than controls. A positive correlation was observed between TNF-a from monocytes of patients and the relevant serum levels (r = 0.58; P < 0.01); (e) conditioned media of lymphocytes and monocytes from patients significantly increased rate of endothelial apoptosis. Conclusions In severe CHF, irrespectively from aetiology, significant activation of immune system occurs: several pro-inflammatory cytokines and soluble factors are spontaneously released in serum, possibly contributing to disease progression, as they induce apoptosis.

Myocardial ischaemia/reperfusion induces NF-kappaB activation, but little is known about the stimuli through which it occurs. Aims of the study were to investigate whether: (a) oxidative stress induced by ischaemia/reperfusion is linked with NF-kappaB activation; (b) counteraction of oxidative stress by N-acetyl cysteine (NAC) reduces NF-kappaB activation. At this purpose, in isolated rat hearts, we induced mild (15 min) and severe (30 min) ischaemia; a group of the hearts submitted to severe ischaemia were treated with NAC. Our data indicate that reperfusion after severe ischaemia activates NF-kappaB: the presence of p65 in the nuclear extracts was 274.5+/-18.6% vs aerobia; (P0.05) and an induced DNA-binding activity was detected. NF-kappaB translocation occurs in parallel with myocardial decrease in reduced glutathione and protein -SH (from 9.2+/-0.4 to 5.4+/-0.3 nmol/mg prot, P0.01, and from 350.3+/-16.6 to 296.0+/-9.1 nmol/mg prot, P0.05) and accumulation of oxidised glutathione-GSSG-(from 0.075+/-0.005 to 0.118+/-0.007 nmol/mg prot, P0.01). When ischaemia/reperfusion does not result in any oxidative stress (in mild ischaemia or severe ischaemia plus NAC), NF-kappaB does not translocate. A significant correlation was found between the activation of NF-kappaB and the accumulation of GSSG in the myocardium. Our data indicate that an oxidative shift of cellular thiolic pools can modulate the genic transcription of the heart through NF-kappaB activation.

Background and aims We recently demonstrated that injection of conditioned medium (CM) generated from cells of the mesenchymal region of human amniotic membrane (AMTCs) reduces bleomycin-induced lung fibrosis in mice, suggesting a crucial role of paracrine factor(s) secreted by AMTCs in these beneficial effects. We further investigated this hypothesis, the mechanisms involved, the effects on some lung functional parameters and whether AMTC-secreted effector(s) are specific to these cells and not produced by other cell types, extending the time of analysis up to 28 days after treatment. Methods Bleomycin-challenged mice were either treated with AMTC-CM or CM generated from human skin fibroblasts, human peripheral blood mononuclear cells or Jurkat cells, or were left untreated. Mouse lungs were analyzed for content of pro-inflammatory and pro-fibrotic molecules, presence of lymphocytes and macrophages and for fibrosis level (through histological semi-quantitative evaluation and quantitative measurement of collagen content). Arterial blood gas analysis was also performed. Results Up to 28 days after delivery, AMTC-CM-treated mice developed reduced lung fibrosis with respect to mice treated with other CM types. AMTC-CM-treated mice had comparatively better preservation of blood gas parameters and showed lower lung content of interleukin-6, tumor necrosis factor-α, macrophage inflammatory protein-1α, monocyte chemoattractant protein-1 and transforming growth factor-β associated with reduced lung macrophage levels. Conclusions AMTC-CM prevents lung fibrosis in bleomycin-challenged mice, improving survival and preserving lung functional parameters such as blood gas exchanges. The specificity of AMTC-CM action was indicated by the absence of fibrosis reduction when other CM types were used. Finally, we provide some insights into the possible mechanisms underlying AMTC-CM-mediated control of fibrosis.

The beta-adrenergic pathway may have a role in the pathophysiology of ischemic syndromes characterised by reversible left ventricular dysfunction, such as myocardial stunning and other clinical conditions of unstable angina or coronary spasms, or chronic reversible left ventricular dysfunction, which might be a consequence of repeated events of short-term ischemia ("repetitive stunning"). A partial-to-total occlusion of the left anterior descending coronary artery in pigs was used to induce short periods of ischemia (total ischemic time 12 +/- 2 min). Hypokinesis and dyskinesis of the myocardium were considered signs of myocardial dysfunction. We found a maintained function of the beta-adrenergic signalling system. Density and affinity of beta-adrenergic receptors were not different in stunned and non-ischemic regions, nor were cyclic AMP and cyclic GMP intracellular contents and ratio, nor well as the ratio of stimulatory/inhibitory G protein a subunits. Our findings are in agreement with a maintained beta-adrenergic signalling system in the pathophysiology of chronic reversible left ventricular dysfunction.

Angiotensin converting enzyme (ACE) inhibitors are under study in ischaemic heart diseases, their mechanism of action being still unknown. The anti-ischaemic effect of trandolapril and the possible involvement of a bradykinin-modulation on endothelial constitutive nitric oxide synthase (eNOS) in exerting this effect, were investigated. Three doses of trandolapril, chronically administered in vivo, were studied in isolated perfused rat hearts subjected to global ischaemia followed by reperfusion. Trandolapril has an anti-ischaemic effect. The dose of 0.3 mg kg−1 exerted the best effect reducing diastolic pressure increase during ischaemia (from 33.0±4.5 to 14.0±5.2 mmHg; P

Angiotensin-converting enzyme (ACE) inhibitors exert some cardiovascular benefits by improving endothelial function. We evaluated the effects of chronic treatment with quinapril (Q) on the l -arginine/nitric oxide (NO) pathway in normotensive rats under baseline and inflammatory conditions. The role of bradykinin was also investigated. The animals received for 1 week either the ACE-inhibitor Q (1 and 10 mg/kg/day), the B(2)receptor antagonist HOE 140, Q+HOE 140, or no drug. At the end of chronic treatment, rats underwent either a 6-h placebo or an E. coli endotoxin challenge. The following measurements were made: (i) endothelial and inducible NO synthase (eNOS and iNOS) protein expression; (ii) eNOS/iNOS activity; (iii) serum levels of nitrite/nitrate and tumour necrosis factor (TNF)- alpha; (iv) NO in the expired air (eNO). Q increased baseline aortic eNOS protein expression (up to 99%, P

Adenosine protects myocardium from ischemia and reperfusion damage; however, the mechanism of action is still under discussion. We investigated whether (a) adenosine protects isolated crystalloid-perfused rabbit heart from ischemia/ reperfusion injury; (b) this action is receptor mediated and what receptor subtypes are involved, and (c) this action is dependent on an enhanced nitric oxide production. Our results showed a cardioprotective effect of adenosine (10(-4) M), of nonselective adenosine-receptor agonist 5'-N-ethyl-carboxamidoadenosine (NECA; 5 x 10(-6) M), and of A2A agonists CGS 21680 (10(-8) and 10(-6) M), 2-hexynylNECA (10(-7) M). On the contrary, A1 agonist CCPA (10(-8) and 10(-6) M) does not provide any protection. The effect has been achieved in terms of significant reduction in contracture development during reperfusion [diastolic pressure was 46.8 +/- 7.1 mm Hg (p < 0.01); 46.1 +/- 7.8 mm Hg (p < 0.01); 46.9 +/- 5.5 mm Hg (p < 0.01); and 59.3 +/- 6.7 mm Hg (p < 0.05) with 10(-4) M adenosine, 5 x 10(-6) M NECA, 10(-6) M CGS 21680, and 10(-7) M 2-hexynylNECA, respectively, versus 77.6 +/- 5.0 mm Hg in control]; reduced creatine phosphokinase release (13.5 +/- 1.6, 22.2 +/- 7.9, 14.2 +/- 3.3, and 14.1 +/- 4.5 U/gww in treated hearts vs. 34.6 +/- 7.2 U/gww in controls; p < 0.05); improved energy metabolism [adenosine triphosphate (ATP) content is 9.9 +/- 0.5, 10.4 +/- 0.6, 9.8 +/- 0.5, and 10.5 +/- 0.5 micromol/gdw in treated hearts vs. 7.6 +/- 0.2 micromol/gdw; p < 0.05]. Moreover, our data indirectly show a functional presence of A2A receptors on cardiomyocytes as the protection is A2A mediated and exerted only during reperfusion, although in the absence of blood and coronary flow changes. These activities appear independent of nitric oxide pathways, as adenosine and 2-hexynylNECA effects are not affected by the presence of a nitric oxide-synthase inhibitor (10(-4) M L-NNA).

Ventricular dysfunction due to myocardial infarction is a result of a pathophysiological “continuum”, proceeding from a fully reversible alteration to the irreversible loss of myocardial tissue. Various studies have suggested that oxidative stress induced by acute ischemia may play a role for the progression of ventricular dysfunction to heart failure. Oxidative injury on the myocardium may be described as the result of two processes; a) a deficit of the endogenous anti-oxidant defence during ischemia, and b) a production of oxygen free radicals occurring during early reperfusion. Ventricular dysfunction may result from a direct action of oxygen free radicals on ion translocating proteins, particularly vulnerable to radical-induced oxidation at key sulfhydryl groups, and an indirect calcium-induced activation of proteases, leading to excitation-contraction uncoupling. A progressive increase in oxygen free radical injury and involvement of oxidative stress have been observed in the evolution of ventricular dysfunction, suggesting that oxidative stress may be an important determinant factor for prognosis. The specific role of oxidative stress in the progression of ischemia to failure is yet to be entirely defined; however, new therapeutic approaches may possible influence the course of this progression by counteracting the oxidative injury.