Monica Forni

Genetically modified pigs produced with a nonviral episomal vector

Genetic modification of cells and animals is an invaluable tool for biotechnology and biomedicine. Currently, integrating vectors are used for this purpose. These vectors, however, may lead to insertional mutagenesis and variable transgene expression and can undergo silencing. Scaffold/matrix attachment region-based vectors are nonviral expression systems that replicate autonomously in mammalian cells, thereby making possible safe and reliable genetic modification of higher eukaryotic cells and organisms. In this study, genetically modified pig fetuses were produced with the scaffold/matrix attachment region-based vector pEPI, delivered to embryos by the sperm-mediated gene transfer method. The pEPI vector was detected in 12 of 18 fetuses in the different tissues analyzed and was shown to be retained as an episome. The reporter gene encoded by the pEPI vector was expressed in 9 of 12 genetically modified fetuses. In positive animals, all tissues analyzed expressed the reporter gene; moreover in these tissues, the positive cells were on the average 79%. The high percentage of EGFP-expressing cells and the absence of mosaicism have important implications for biotechnological and biomedical applications. These results are an important step forward in animal transgenesis and can provide the basis for the future development of germ-line gene therapy.

Carbon monoxide improves cardiac energetics and safeguards the heart during reperfusion after cardiopulmonary bypass in pigs

Ischemia‐reperfusion injury, a clinical problem during cardiac surgery, involves worsened adenosine trisphosphate (ATP) generation and damage to the heart. We studied carbon monoxide (CO) pretreatment, proven valuable in rodents but not previously tested in large animals, for its effects on pig hearts subjected to cardiopulmonary bypass with cardioplegic arrest. Hearts of CO‐treated pigs showed significantly higher ATP and phosphocreatine levels, less interstitial edema, and apoptosis of cardiomyocytes and required fewer defibrillations after bypass. We conclude that treatment with CO improves the energy status, prevents edema formation and apoptosis, and facilitates recovery in a clinically relevant model of cardiopulmonary bypass surgery.

Carbon monoxide pretreatment prevents respiratory derangement and ameliorates hyperacute endotoxic shock in pigs

Endotoxic shock, one of the most prominent causes of mortality in intensive care units, is characterized by pulmonary hypertension, systemic hypotension, heart failure, widespread endothelial activation/injury, and clotting culminating in disseminated intravascular coagulation and multi‐organ system failure. In the last few years, studies in rodents have shown that administration of low concentrations of carbon monoxide (CO) exerts potent therapeutic effects in a variety of diseases/disorders. In this study, we have administered CO (one our pretreatment at 250 ppm) in a clinically relevant, well‐characterized model of LPS‐induced acute lung injury in pigs. Pretreatment only with inhaled CO significantly ameliorated several of the acute pathological changes induced by endotoxic shock. In terms of lung physiology, CO pretreatment corrected the LPS‐induced changes in resistance and compliance and improved the derangement in pulmonary gas exchange. In terms of coagulation and inflammation, CO reduced the development of disseminated intravascular coagulation and completely suppressed serum levels of the proinflammatory IL‐1β in response to LPS, while augmenting the anti‐inflammatory cytokine IL‐10. Moreover, the effects of CO blunted the deterioration of kidney and liver function, suggesting a beneficial effect in terms of end organ damage associated with endotoxic shock. Lastly, CO pretreatment prevents LPS‐induced ICAM expression on lung endothelium and inhibits leukocyte marginalization on lung parenchyma.

In situ detection of apoptosis in regressing corpus luteum of pregnant sow: Evidence of an early presence of DNA fragmentation

Luteolysis has been shown to be correlated with apoptosis in rats, sheep, and cows. In pigs, apoptosis has already been demonstrated as regards atretic follicles. The present study has been conducted to evaluate whether apoptosis occurs during corpora lutea regression in the pregnant pig and to investigate the temporal relationship between apoptosis and functional luteolysis. The apoptotic process has been studied through the research of oligonucleosome fragmentation by means of classical electrophoresis methods and by in situ detection on histological luteal sections. The latter method allows the identification of apoptosis and the localization of apoptotic cells. Pregnant sows were cloprostenol (PGF2 alpha analog) treated and ovariectomized 0, 6, 12, 24, 48, and 72 hr after treatment. Corpora lutea were utilized for progesterone and DNA extraction and in situ evaluation of apoptosis. Clear evidence of apoptosis was seen earlier with the in situ technique (6 hr for stromal tissue, 12 hr for luteal cells) than with the classical method (24 hr). Apoptosis was, however, apparent after plasma and tissue progesterone had reached basal levels. In conclusion, these results are consistent with the hypothesis that apoptosis occurs during luteolysis in pigs. Moreover, the data obtained with the in situ technique made it possible to identify signs of structural regression in stromal tissue first than in parenchymal cells. A two-stage activation of apoptosis has been discussed to explain structural changes that occur during luteolysis after cloprostenol treatment in swine corpora lutea.

Expression of endothelin-1 system in a pig model of endotoxic shock

Endothelin (ET)-1 is a potent vasoconstrictive peptide and it is involved in the pathogenesis of septic shock. Blockade of ET-1 receptors abolishes the LPS-induced pulmonary hypertension and worsens the LPS-dependent systemic hypotension, but the role of ET-1 in sepsis remains uncertain. To determine the role of ET-1 in cardiovascular and respiratory derangement in a porcine model of endotoxemic shock we evaluated ET-1 plasma levels and ET-1 mRNA and protein levels in lung, liver, and heart as well as Endothelin Converting Enzyme-1, ET(A) and ET(B) receptors mRNA in the same tissues. Twelve piglets were randomised to sham operated or to LPS-treated (40 microg/kg/h for 4 h) groups. During the experiment, respiratory and circulatory parameters have been recorded and blood samples collected. At the end of the experiment the animals were sacrificed and tissue samples collected for real-time quantitative PCR and ELISA test. LPS infusion evokes a large increase in ET-1 plasma concentration, and in tissues mRNA levels, associated with an increase in pulmonary arterial pressure, as well as in pulmonary and systemic vascular resistances, and a decrease in stroke volume. LPS infusion caused also a derangement of respiratory mechanics, evidenced by an increase in resistance and a decrease in compliance of the respiratory system. ET(A) and ET(B) receptor mRNA levels were markedly decreased in liver and lung and slightly increased in heart, evidencing that ET receptor subtypes were differentially regulated in the major organs of endotoxin treated pigs. In conclusion our data show the presence of a continuative and differentially regulated stimulating mechanism of ET-1 expression during pig endotoxaemia as well as a fundamental role of ET-1 system in the cardiovascular and respiratory derangement.

Heat shock protein 70, heat shock protein 32, and vascular endothelial growth factor production and their effects on lipopolysaccharide-induced apoptosis in porcine aortic endothelial cells

Abstract Lipopolysaccharide (LPS) is a highly proactive molecule that causes in vivo a systemic inflammatory response syndrome and activates in vitro the inflammatory pathway in different cellular types, including endothelial cells (EC). Because the proinflammatory status could lead to EC injury and apoptosis, the expression of proinflammatory genes must be finely regulated through the induction of protective genes. This study aimed at determining whether an LPS exposure is effective in inducing apoptosis in primary cultures of porcine aortic endothelial cells and in stimulating heat shock protein (Hsp)70 and Hsp32 production as well as vascular endothelial growth factor (VEGF) secretion. Cells between third and eighth passage were exposed to 10 μg/mL LPS for 1, 7, 15, and 24 hours (time-course experiments) or to 1, 10, and 100 μg/mL LPS for 7 and 15 hours (dose-response experiments). Apoptosis was not affected by 1 μg/mL LPS but significantly increased in a dose-dependent manner with the highest LPS doses. Furthermore, apoptosis rate increased only till 15 hours of LPS exposure. LPS stimulated VEGF secretion in a dose-dependent manner; its effect became significant after 7 hours and reached a plateau after 15 hours. Both Hsp70 and Hsp32 expressions were induced by LPS in a dose-dependent manner after 7 hours. Subsequent studies were addressed to evaluate the protective role of Hsp32, Hsp70, and VEGF. Hemin, an Hsp32 inducer (5, 20, 50 μM), and recombinant VEGF (100 and 200 ng/mL), were added to the culture 2 hours before LPS (10 μg/mL for 24 hours); to induce Hsp70 expression, cells were heat shocked (42°C for 1 hour) 15 hours before LPS (10 μg/mL for 24 hours). Hemin exposure upregulated Hsp32 expression in a dose-dependent manner and protected cells against LPS-induced apoptosis. Heat shock (HS) stimulated Hsp70 expression but failed to reduce LPS-induced apoptosis; VEGF addition did not protect cells against LPS-induced apoptosis at any dose tested. Nevertheless, when treatments were associated, a reduction of LPS-induced apoptosis was always observed; the reduction was maximal when all the treatments (HS + Hemin + VEGF) were associated. In conclusion, this study demonstrates that LPS is effective in evoking “the heat shock response” with an increase of nonspecific protective molecules (namely Hsp70 and Hsp32) and of VEGF, a specific EC growth factor. The protective role of Hsp32 was also demonstrated. Further investigations are required to clarify the synergic effect of Hsp32, Hsp70, and VEGF, thus elucidating the possible interaction between these molecules.

Gelatinases, endonuclease and Vascular Endothelial Growth Factor during development and regression of swine luteal tissue

BackgroundThe development and regression of corpus luteum (CL) is characterized by an intense angiogenesis and angioregression accompanied by luteal tissue and extracellular matrix (ECM) remodelling. Vascular Endothelial Growth Factor (VEGF) is the main regulator of angiogenesis, promoting endothelial cell mitosis and differentiation. After the formation of neovascular tubes, the remodelling of ECM is essential for the correct development of CL, particularly by the action of specific class of proteolytic enzymes known as matrix metalloproteinases (MMPs). During luteal regression, characterized by an apoptotic process and successively by an intense ECM and luteal degradation, the activation of Ca++/Mg++-dependent endonucleases and MMPs activity are required. The levels of expression and activity of VEGF, MMP-2 and -9, and Ca++/Mg++-dependent endonucleases throughout the oestrous cycle and at pregnancy were analyzed.ResultsDifferent patterns of VEGF, MMPs and Ca++/Mg++-dependent endonuclease were observed in swine CL during different luteal phases and at pregnancy. Immediately after ovulation, the highest levels of VEGF mRNA/protein and MMP-9 activity were detected. On days 5–14 after ovulation, VEGF expression and MMP-2 and -9 activities are at basal levels, while Ca++/Mg++-dependent endonuclease levels increased significantly in relation to day 1. Only at luteolysis (day 17), Ca++/Mg++-dependent endonuclease and MMP-2 spontaneous activity increased significantly. At pregnancy, high levels of MMP-9 and VEGF were observed.ConclusionOur findings, obtained from a precisely controlled in vivo model of CL development and regression, allow us to determine relationships among VEGF, MMPs and endonucleases during angiogenesis and angioregression. Thus, CL provides a very interesting model for studying factors involved in vascular remodelling.

Inhaled Carbon Monoxide (CO) Prevents Lung Oedema Induced by Endotoxic Shock

S. Mazzola1, M. Forni2, M. Albertini1*, M.L. Bacci2, B. Ciminaghi1, M. Lavitrano3, E. Seren2 and M.G. Clement1 1Department of Animal Pathology, Hygiene and Public Veterinary Health, Section of Biochemistry and Physiology, University of Milan, Italy; 2Department of Veterinary Morphophysiology and Animal Production, University of Bologna, Italy; 3Department of Experimental Environmental Medicine and Medical Biotechnology, University of Milano-Bicocca, Italy *Correspondence: Dipartimento di Patologia Animale, Igiene e Sanita Pubblica Veterinaria – Sezione di Biochimica e Fisiologia Veterinaria – Facolta di Medicina Veterinaria – Universita degli Studi di Milano, V ia Celoria 10, 20133 Milano, Italy E-mail: mariangela.albertini@unimi.it

Induced metastable memory in heat shock response.

We studied the dynamics of the Heat Shock Response (HSR) mechanism, and the persistence of a injury-protected state in the cell following the shocks, known as thermotolerance. A series of double shock experiments were performed on Chinese Hamster Ovary (CHO) cells, tracking the dynamics of some components of HSR pathway (the Hsp70 protein level and Hsp70 mRNA transcription rate). The main features of HSR dynamics were well reproduced by a simplified model of the chemical reaction pathways governing the HSR. In particular, the thermotolerance phenomenon could be well characterized by introducing a shock-dependent switch in mRNA halflife, that can be interpreted as a sort of primitive memory at the mRNA level.

Differential expression of VEGF isoforms in pig corpora lutea throughout the estrous cycle

Angiogenesis is a key process of dynamic modification that takes place in the cyclic ovarian function. In particular, many studies have demonstrated that the regulation of angiogenesis is fundamental to a correct development of the ovarian glandular structure (Ferrara et al., 2003). Among the molecules involved in angiogenesis, VEGF is the most important. Five VEGF isoforms are produced by alternative splicing of the same gene, two of them (VEGF121 and VEGF165) are secreted and may act as paracrine/endocrine, whereas the others (VEGF145, VEGF189 and VEGF206) are mostly cell associated and may act as autocrine. In the majority of the studied species, two principal VEGF transcripts were identified in the corpus luteum, VEGF121 and VEGF165 (Redmer et al., 1996; Berisha et al., 2000; Sugino et al., 2000; Barboni et al., 2000); VEGF189 was also observed in ovine corpus luteum (Redmer et al., 1996). Recently, it was demonstrated that the expression level of VEGF165 mRNA in the swine corpus luteum was constant during the estrous cycle (Boonyaprakob et al., 2003). This study was aimed at identifying the different VEGF mRNA isoforms in the swine corpus luteum and to evaluate the variation in their expression levels throughout the estrous cycle by real time RT-PCR, a quantitative and high sensitive technique.