In vivo inhibition of rat stellate cell activation by soluble transforming growth factor β type II receptor: A potential new therapy for hepatic fibrosis

Transforming growth factor β (TGF-β) is a well characterized cytokine that appears to play a major role in directing the cellular response to injury, driving fibrogenesis, and, thus, potentially underlying the progression of chronic injury to fibrosis. In this study, we report the use of a novel TGF-β receptor antagonist to block fibrogenesis induced by ligation of the common bile duct in rats. The antagonist consisted of a chimeric IgG containing the extracellular portion of the TGF-β type II receptor. This “soluble receptor” was infused at the time of injury; in some experiments it was given at 4 days after injury, as a test of its ability to reverse fibrogenesis. The latter was assessed by expression of collagen, both as the mRNA in stellate cells isolated from control or injured liver and also by quantitative histochemistry of tissue sections. When the soluble receptor was administered at the time of injury, collagen I mRNA in stellate cells from the injured liver was 26% of that from animals receiving control IgG (P < 0.0002); when soluble receptor was given after injury induction, collagen I expression was 35% of that in control stellate cells (P < 0.0001). By quantitative histochemistry, hepatic fibrosis in treated animals was 55% of that in controls. We conclude that soluble TGF-β receptor is an effective inhibitor of experimental fibrogenesis in vivo and merits clinical evaluation as a novel agent for controlling hepatic fibrosis in chronic liver injury.

Angiogenesis promoted by vascular endothelial growth factor: Regulation through α1β1 and α2β1 integrins

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a cytokine of central importance for the angiogenesis associated with cancers and other pathologies. Because angiogenesis often involves endothelial cell (EC) migration and proliferation within a collagen-rich extracellular matrix, we investigated the possibility that VEGF promotes neovascularization through regulation of collagen receptor expression. VEGF induced a 5- to 7-fold increase in dermal microvascular EC surface protein expression of two collagen receptors—the α1β1 and α2β1 integrins—through induction of mRNAs encoding the α1 and α2 subunits. In contrast, VEGF did not induce increased expression of the α3β1 integrin, which also has been implicated in collagen binding. Integrin α1-blocking and α2-blocking antibodies (Ab) each partially inhibited attachment of microvascular EC to collagen I, and α1-blocking Ab also inhibited attachment to collagen IV and laminin-1. Induction of α1β1 and α2β1 expression by VEGF promoted cell spreading on collagen I gels which was abolished by a combination of α1-blocking and α2-blocking Abs. In vivo, a combination of α1-blocking and α2-blocking Abs markedly inhibited VEGF-driven angiogenesis; average cross-sectional area of individual new blood vessels was reduced 90% and average total new vascular area was reduced 82% without detectable effects on the pre-existing vasculature. These data indicate that induction of α1β1 and α2β1 expression by EC is an important mechanism by which VEGF promotes angiogenesis and that α1β1 and α2β1 antagonists may prove effective in inhibiting VEGF-driven angiogenesis in cancers and other important pathologies.

Skin wounds and severed nerves heal normally in mice lacking tenascin-C.

A large number of functions have been demonstrated for tenascin-C by antibody perturbation assays and in vitro cell culture experiments. However, these results contrast sharply with the lack of any apparent phenotype in mice with a genetic deletion of tenascin-C. A possible explanation for the lack of phenotype would be expression of some altered but functional tenascin-C in the mutant. We report the generation of an independent tenascin-C null mouse and conclude that the original tenascin-C knockout, which is genetically very similar to ours, is also a true null. As found previously, the absence of tenascin-C has no influence on development, adulthood, life span, and fecundity. We have studied in detail two models of wound healing. After axotomy, the regeneration of the sciatic nerve is not altered without tenascin-C. During healing of cutaneous wounds, deposition of collagen I, fibulin-2, and nidogen is identical in mutant and wild-type mice. In contrast. fibronectin appears diminished in wounds of tenascin-C-deficient mice. However, the lack of tenascin-C together with the reduced amount of fibronectin has no influence on the quality of the healing process.

Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice.

Cells from transgenic mice expressing a human mini-gene for collagen I were used as markers to follow the fate of mesenchymal precursor cells from marrow that were partially enriched by adherence to plastic, expanded in culture, and then injected into irradiated mice. Sensitive PCR assays for the marker collagen I gene indicated that few of the donor cells were present in the recipient mice after 1 week, but 1-5 months later, the donor cells accounted for 1.5-12% of the cells in bone, cartilage, and lung in addition to marrow and spleen. A PCR in situ assay on lung indicated that the donor cells diffusely populated the parenchyma, and reverse transcription-PCR assays indicated that the marker collagen I gene was expressed in a tissue-specific manner. The results, therefore, demonstrated that mesenchymal precursor cells from marrow that are expanded in culture can serve as long-lasting precursors for mesenchymal cells in bone, cartilage, and lung. They suggest that cells may be particularly attractive targets for gene therapy ex vivo.

Differential tubulogenic and branching morphogenetic activities of growth factors: implications for epithelial tissue development.

At least two kidney epithelial cell lines, the Madin-Darby canine kidney (MDCK) and the murine inner medullary collecting duct line mIMCD-3, can be induced to form branching tubular structures when cultured with hepatocyte growth factor (HGF) plus serum in collagen I gels. In our studies, whereas MDCK cells remained unable to form tubules in the presence of serum alone, mIMCD-3 cells formed impressive branching tubular structures with apparent lumens, suggesting the existence of specific factors in serum that are tubulogenic for mIMCD-3 cells but not for MDCK cells. Since normal serum does not contain enough HGF to induce tubulogenesis, these factors appeared to be substances other than HGF. This was also suggested by another observation: when MDCK cells or mIMCD-3 cells were cocultured under serum-free conditions with the embryonic kidney, both cell types formed branching tubular structures similar to those induced by HGF; however, only in the case of MDCK cells could this be inhibited by neutralizing antibodies against HGF. Thus, the embryonic kidney produces growth factors other than HGF capable of inducing tubule formation in the mIMCD-3 cells. Of a number of growth factors examined, transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) were found to be tubulogenic for mIMCD-3 cells. Whereas only HGF was a potent tubulogenic factor for MDCK cells, HGF, TGF-alpha, and EGF were potent tubulogenic factors for mIMCD-3 cells. Nevertheless, there were marked differences in the capacity of these tubulogenic factors to induce tubulation as well as branching events in those tubules that did form (HGF >> TGF-alpha > EGF). Thus, at least three different growth factors can induce tubulogenesis and branching in a specific epithelial cell in vitro (though to different degrees), and different epithelial cells that are capable of forming branching tubular structures demonstrate vastly different responses to tubulogenic growth factors. The results are discussed in the context of branching morphogenesis during epithelial tissue development.

Propriedades de soluções filmogênicas e de filmes de gelatina ou colágeno com extrato de boldo-do-Chile

Filmes à base de biopolímeros podem ser utilizados para produção de embalagens ativas, que além de proteger os alimentos, podem interagir com o produto. No caso de embalagens ativas com atividade antioxidante, tem-se privilegiado o uso de agentes antioxidantes naturais, considerando-se que o uso de antioxidantes sintéticos tem sido questionado, sobretudo em relação à sua inocuidade. Existem muitos extratos de plantas já conhecidos por sua atividade antioxidante, que têm sido utilizados com frequência em estudos de filmes à base de biopolímeros, não somente por serem ricos em polifenóis, mas principalmente por sua boa interação com a matriz polimérica. O extrato de boldo-do-Chile (Peumus boldus) possui atividade antioxidante comprovada, entretanto, não existem relatos sobre sua adição em filmes. Dessa forma, o objetivo geral desta tese foi o desenvolvimento de filmes à base de colágeno ou gelatina adicionados de extrato de boldo-do-Chile, com propriedades físicas e funcionais para seu emprego como embalagens bioativas. Foram realizadas as caracterizações (fenólicos totais, ABTS, DPPH, cor, °Brix e pH) do extrato aquoso de boldo-do-Chile, preparado em quatro diferentes temperaturas. Além disso, foram avaliadas as propriedades reológicas e térmicas da solução de gelatina, e também foram elaborados filmes com as soluções de gelatina e colágeno a partir de soluções filmogênicas com diferentes concentrações de macromoléculas e extrato de boldo. Esses filmes foram caracterizados para conhecimento de suas propriedades mecânicas (tração e perfuração), propriedades óticas (cor e opacidade), espessura, umidade e solubilidade em água. Uma concentração de macromoléculas foi escolhida para a realização de análises complementares, a saber: análises térmicas (DSC), cristalinidade por difração de raio X (DRX), permeabilidade ao vapor de água (PVA), microscopia eletrônica de varredura (MEV), brilho, espectroscopia de infravermelho com transformada de Fourier (FTIR), ângulo de contato, propriedades de barreira UV/Visível e atividade antioxidante. A adição do extrato de boldo-do-Chile nos filmes de gelatina e colágeno produziu filmes com atividade antioxidante, sem prejuízo às demais propriedades estudadas. Observou-se que o extrato aquoso de boldo-do-Chile apresentou propriedades antioxidantes, mas que foram dependentes da temperatura de extração. O extrato de boldo-do-Chile foi capaz de modificar as propriedades térmicas das soluções filmogênicas de gelatina, não sendo observado efeito nas análises reológicas. Por outro lado, o extrato aquoso de boldo-do-Chile não influenciou as propriedades mecânicas, solubilidade, umidade, cristalinidade e a permeabilidade ao vapor de água dos filmes de gelatina ou colágeno. Algumas propriedades térmicas sofreram efeito dos extratos, mas as análises de FTIR não mostraram a formação de novas interações. As propriedades óticas e de barreira UV/Visível foram influenciadas pelo extrato de boldo-do-Chile, sendo que os filmes se apresentaram mais amarelados com o aumento da concentração do extrato de boldo-do-Chile. As micrografias mostraram filmes de gelatina bastante homogêneos e filmes de colágeno com superfícies mais rugosas. Os resultados de brilho e ângulo de contato corroboraram com estas respostas. Em conclusão, os filmes de ambas macromoléculas apresentaram atividade antioxidante, podendo dessa forma, serem considerados como filmes ativos.

Tumor progression in hepatocellular carcinoma: Relationship with tumor stroma and parenchymal disease

Background: Encapsulation in hepatocellular carcinoma is associated with decreased invasiveness and improved survival in several series. Although active fibrogenesis by myofibroblasts has been demonstrated in the capsule, it is unclear if the capsule results from a general increase in peritumoral fibrosis, or an inherently less invasive tumor phenotype. The relationship between collagen deposition within tumor stroma, presence of cirrhosis and invasiveness also needs clarification. Methods: We performed immunohistochemistry for collagens I, III, IV and VI on sections of encapsulated and non-encapsulated hepatocellular carcinoma, arising in cirrhotic and non-cirrhotic livers. Staining was graded semi-quantitatively in tumor stromal elements and adjacent parenchymal sinusoids. The relationship of this staining with encapsulation, cirrhosis, and vascular invasion was analyzed. Results: Formation of a discrete capsular layer was associated with reduced vascular invasion, but not with a pervasive increase in peritumoral fibrosis. Increased collagen I content of tumor stroma and adjacent parenchymal sinusoids was associated with non-encapsulated tumors and vascular invasion. The presence of cirrhosis had little effect on capsule composition. Conclusions: Encapsulation of hepatocellular carcinoma reflects reduced invasiveness, rather than increased peritumoral collagen synthesis, which may instead enhance invasion. Increased intratumoral collagen I protein is also associated with increased tumor invasiveness. Pre-existing cirrhosis has little effect on tumor progression, possibly because the characteristics of cirrhosis are overwhelmed by tumor-induced changes in the adjacent parenchyma.(C) 2003 Blackwell Publishing Asia Pty Ltd.

Transglutaminase inhibition reduces fibrosis and preserves function in experimental chronic kidney disease

Progressive tissue fibrosis is involved in debilitating diseases that affect organs including the lungs, liver, heart, skin, and kidneys. Recent evidence suggests that tissue transglutaminase, an enzyme that crosslinks proteins, may be involved in tissue fibrosis by crosslinking and stabilizing the extracellular matrix or by recruiting and activating the large latent transforming growth factor (TGF)-β1 complex. We treated rats that had undergone 5/6-nephrectomy with two different irreversible inhibitors of transglutaminase and found that both prevented a decline in kidney function and reduced the development of glomerulosclerosis and tubulointerstitial fibrosis by up to 77% and 92%, respectively. Treatment reduced the accumulation of collagen I and collagen III, with the primary mechanism of action being direct interference with the crosslinking of extracellular matrix rather than altered regulation of TGFβ1. We conclude that inhibition of transglutaminase offers a potential therapeutic option for chronic kidney disease and other conditions that result from tissue fibrosis. Copyright © 2007 by the American Society of Nephrology.

Modelling the gastric epithelium for testing of new chemical entities

A cell culture model of the gastric epithelial cell surface would prove useful for biopharmaceutical screening of new chemical entities and dosage forms. A successful model should exhibit tight junction formation, maintenance of differentiation and polarity. Conditions for primary culture of guinea-pig gastric mucous epithelial cell monolayers on Tissue Culture Plastic (TCP) and membrane insects (Transwells) were established. Tight junction formation for cells grown on Transwells for three days was assessed by measurement of transepithelial resistance (TEER) and permeability of mannitol and fluorescein. Coating the polycarbonate filter with collagen IV, rather with collagen I, enhanced tight junction formation. TEER for cells grown on Transwells coated with collagen IV was close to that obtained with intact guinea-pig gastric epithelium in vitro. Differentiation was assessed by incorporation of [3H] glucosamine into glycoprotein and by activity of NADPH oxidase, which produces superoxide. Both of these measures were greater for cells grown on filters coated with collagen I than for cells grown on TCP, but no major difference was found between cells grown on collagens I and IV. However, monolayers grown on membranes coated with collagen IV exhibited apically polarized secretion of mucin and superoxide. The proportion of cells, which stained positively for mucin with periodic Schiff reagent, was greater than 95% for all culture conditions. Gastric epithelial monolayers grown on Transwells coated with collagen IV were able to withstand transient (30 min) apical acidification to pH 3, which was associated with a decrease in [3H] mannitol flux and an increase in TEER relative to pH 7.4. The model was used to provide the first direct demonstration that an NSAID (indomethacin) accumulated in gastric epithelial cells exposed to low apical pH. In conclusion, guinea-pig epithelial cells cultured on collagen IV represent a promising model of the gastric surface epithelium suitable for screening procedures.

TG2: a credible therapeutic target in fibrotic diseasedue to its multifunctional roles

The role of TG2 in fibrosis is reported to be related to two important effects. The first in mediating the deposition and accumulation of the fibrotic extracellular matrix (ECM) via its cross-linking of proteins like fibronectin, collagen I and collagen III; and the second, the activation of latent matrix bound TGFβ1. We report here that the role of TG2 in fibrosis progression can be much more complex. We also report a new family of TG2-specific inhibitors that can not only inhibit protein cross-linking, but also regulate other functions of TG2, thus increasing their potency which can be demonstrated by their effectiveness in inhibiting fibrosis in two different fibrotic in vivo models.

Efeitos da terapia laser de baixa intensidade associada ao exercício aeróbio no reparo de tenotomia do tendão de aquiles de ratos diabéticos

Diabetes Mellitus (DM ) is a complex disease that requires continuous medical care for the reduction of risk factors in addition to glycemic control. The typical hyperglycemia of this disease produces glycosylation of proteins and so the consequence is the accumulation of glycosylation final products in various human tissues, among them, the tendon. The aerobic exercise (AE) and the low level laser therapy (LLLT) have been used to treat tendinopathies in individuals with or without DM. Objective: The aim of this study was to watch the effect of the LLLT and the AE, in association, in partial tenotomy of the tissue repair of the Achilles tendon (AT) of diabetic rats. Methods: 91 animals were utilized and divided in to the following groups: control group (GC), injured control group (GCL), diabetic group (GD), diabetic group LLLT (GD – TLBI), diabetic group trained (GD - EX) and diabetic group trained laser (GD-EX+TLBI). The animals were submitted to intervention with AE, using a protocol with a progressive increase of time (12 to 60 min) and speed of (4 to 9 m/min), and the LLLT (660 nm laser, 10mW, 4 J/cm², single point for 16 seconds, three times for week). It was analyzed morphological, biomechanical and molecular characteristics. For data showing normal distribution was used one-way ANOVA test and post hoc Tukey and data without normal distribution was used Mann Whitney test and post hoc Dunn's. It was accepted p <0.05 for statistical significance Results: The biomechanical tests indicated major improvement in the GC and GD-EX+TLBI groups when compared with the diabetic groups in the following variables: maximum load, strain, absorbed energy, stress, cross section area, elastic modulus and energy density (p<0.05). The analysis through molecular biology indicated that the association of aerobic exercise and LLLT generated an increase of the collagen I gene expression and modulated the expression of the MMP2 and MMP9 (p<0.05). No observed any major improvement in the morphological variable studied. Conclusion: the LLLT associated with aerobic exercise promotes and increase of the mechanical properties, in the control of collagen I gene expression and of the MMP2 and MMP9 of the diabetic rats.

Epigenetic Therapy for the Treatment of Hypertension-Induced Cardiac Hypertrophy and Fibrosis

BACKGROUND: The development of heart failure is associated with changes in the size, shape, and structure of the heart that has a negative impact on cardiac function. These pathological changes involve excessive extracellular matrix deposition within the myocardial interstitium and myocyte hypertrophy. Alterations in fibroblast phenotype and myocyte activity are associated with reprogramming of gene transcriptional profiles that likely requires epigenetic alterations in chromatin structure. The aim of our work was to investigate the potential of a currently licensed anticancer epigenetic modifier as a treatment option for cardiac diseases associated with hypertension-induced cardiac hypertrophy and fibrosis.

METHODS AND RESULTS: The effects of DNA methylation inhibition with 5-azacytidine (5-aza) were examined in a human primary fibroblast cell line and in a spontaneously hypertensive rat (SHR) model. The results from this work allude to novel in vivo antifibrotic and antihypertrophic actions of 5-aza. Administration of the DNA methylation inhibitor significantly improved several echocardiographic parameters associated with hypertrophy and diastolic dysfunction. Myocardial collagen levels and myocyte size were reduced in 5-aza-treated SHRs. These findings are supported by beneficial in vitro effects in cardiac fibroblasts. Collagen I, collagen III, and α-smooth muscle actin were reduced in a human ventricular cardiac fibroblast cell line treated with 5-aza.

CONCLUSION: These findings suggest a role for epigenetic modifications in contributing to the profibrotic and hypertrophic changes evident during disease progression. Therapeutic intervention with 5-aza demonstrated favorable effects highlighting the potential use of this epigenetic modifier as a treatment option for cardiac pathologies associated with hypertrophy and fibrosis.

Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype

BACKGROUND: Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression.

METHODS: CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter.

RESULTS: Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2'-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O2.

CONCLUSION: These data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia.

The regulation of cancer cell invasive behaviour by Chloride Interacellular Channel 3 (CLIC3) and Transglutaminasae 2

A study into the role of secreted CLIC3 in tumour cell invasion. The initiation and progression of cancers is thought to be linked to their relationship with a population of activated fibroblasts, which are associated with tumours. I have used an organotypic approach, in which plugs of collagen I are preconditioned with fibroblastic cells, to characterise the mechanisms through which carcinoma-associated fibroblasts (CAFs) influence the invasive behaviour of tumour cells. I have found that immortalised cancer-associated fibroblasts (iCAFs) support increased invasiveness of cancer cells, and that this is associated with the ability of CAFs to increase the fibrillar collagen content of the extracellular matrix (ECM). To gain mechanistic insight into this phenomenon, an in-depth SILAC-based mass proteomic analysis was conducted, which allowed quantitative comparison of the proteomes of iCAFs and immortalised normal fibroblast (iNFs) controls. Chloride Intracellular Channel Protein 3 (CLIC3) was one of the most significantly upregulated components of the iCAF proteome. Knockdown of CLIC3 in iCAFs reduced the ability of these cells to remodel the ECM and to support tumour cell invasion through organotypic plugs. A series of experiments, including proteomic analysis of cell culture medium that had been preconditioned by iCAFs, indicated that CLIC3 itself was a component of the iCAF secretome that was responsible for the ability of iCAFs to drive tumour cell invasiveness. Moreover, addition of soluble recombinant CLIC3 (rCLIC3) was sufficient to drive the extension of invasive pseudopods in cancer cell lines, and to promote disruption of the basement membrane in a 3D in vitro model of the ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) transition. My investigation into the mechanism through which extracellular CLIC3 drives tumour cell invasiveness led me to focus on the relationship between CLIC3 and the ECM modifying enzyme, transglutaminase-2 (TG2). Through this, I have found that TG2 physically associates with CLIC3 and that TG2 is necessary for CLIC3 to drive tumour cell invasiveness. These data identifying CLIC3 as a key pro-invasive factor, which is secreted by CAFs, provides an unprecedented mechanism through which the stroma may drive cancer progression.

Investigating Angiotensin II in cardiac remodelling and the counter-regulatory actions of Angiotensin-(1-9)

Cardiovascular diseases (CVDs) including, hypertension, coronary heart disease and heart failure are the leading cause of death worldwide. Hypertension, a chronic increase in blood pressure above 140/90 mmHg, is the single main contributor to deaths due to heart disease and stroke. In the heart, hypertension results in adaptive cardiac remodelling, including LV hypertrophy to normalize wall stress and maintain cardiac contractile function. However, chronic increases in BP results in the development of hypertensive heart disease (HHD). HHD describes the maladaptive changes during cardiac remodelling which result in reduced systolic and diastolic function and eventually heart failure. This includes ventricular dilation due to eccentric hypertrophy, cardiac fibrosis which stiffens the ventricular wall and microvascular rarefaction resulting in a decrease in coronary blood flow albeit an increase in energy demand. Chronic activation of the renin-angiotensin-system (RAS) with its effector peptide angiotensin (Ang)II plays a key role in the development of hypertension and the maladaptive changes in HHD. Ang II acts via the angiotensin type 1 receptor (AT1R) to mediate most of its pathological actions during HHD, including stimulation of cardiomyocyte hypertrophy, activation of cardiac fibroblasts and increased collagen deposition. The counter-regulatory axis of the RAS which is centred on the ACE2/Ang-(1-7)/Mas axis has been demonstrated to counteract the pathological actions of Ang II in the heart and vasculature. Ang-(1-7) via the Mas receptor prevents Ang II-induced cardiac hypertrophy and fibrosis and improves cardiac contractile function in animal models of HHD. In contrast, less is known about Ang-(1-9) although evidence has demonstrated that Ang-(1-9) also antagonises Ang II and is anti-hypertrophic and anti-fibrotic in animal models of acute cardiac remodelling. However, so far it is not well documented whether Ang-(1-9) can reverse established cardiac dysfunction and remodelling and whether it is beneficial when administered chronically. Therefore, the main aim of this thesis was to assess the effects of chronic Ang-(1-9) administration on cardiac structure and function in a model of Ang II-induced cardiac remodelling. Furthermore, this thesis aimed to investigate novel pathways contributing to the pathological remodelling in response to Ang II. First, a mouse model of chronic Ang II infusion was established and characterised by comparing the structural and functional effects of the infusion of a low and high dose of Ang II after 6 weeks. Echocardiographic measurements demonstrated that low dose Ang II infusion resulted in a gradual decline in cardiac function while a high dose of Ang II induced acute cardiac contractile dysfunction. Both doses equally induced the development of cardiac hypertrophy and cardiac fibrosis characterised by an increase in the deposition of collagen I and collagen III. Moreover, increases in gene expression of fibrotic and hypertrophic markers could be detected following high dose Ang II infusion over 6 weeks. Following this characterisation, the high dose infusion model was used to assess the effects of Ang-(1-9) on cardiac structural and functional remodelling in established disease. Initially, it was evaluated whether Ang-(1-9) can reverse Ang II-induced cardiac disease by administering Ang-(1-9) for 2-4 weeks following an initial 2 week infusion of a high dose of Ang II to induce cardiac contractile dysfunction. The infusion of Ang-(1-9) for 2 weeks was associated with a significant improvement of LV fractional shortening compared to Ang II infusion. However, after 4 weeks fractional shortening declined to Ang II levels. Despite the transient improvement in cardiac contractile function, Ang-(1-9) did not modulate blood pressure, LV hypertrophy or cardiac fibrosis. To further investigate the direct cardiac effects of Ang-(1-9), cardiac contractile performance in response to Ang-(1-9) was evaluated in the isolated Langendorff-perfused rat heart. Perfusion of Ang-(1-9) in the paced and spontaneously beating rat heart mediated a positive inotropic effect characterised by an increase in LV developed pressure, cardiac contractility and relaxation. This was in contrast to Ang II and Ang-(1-7). Furthermore, the positive inotropic effect to Ang-(1-9) was blocked by the AT1R antagonist losartan and the protein kinase A inhibitor H89. Next, endothelial-to-mesenchymal transition (EndMT) as a novel pathway that may contribute to Ang II-induced cardiac remodelling was assessed in Ang II-infused mice in vivo and in human coronary artery endothelial cells (HCAEC) in vitro. Infusion of Ang II to mice for 2-6 weeks resulted in a significant decrease in myocardial capillary density and this was associated with the occurrence of dual labelling of endothelial cells for endothelial and mesenchymal markers. In vitro stimulation of HCAEC with TGFβ and Ang II revealed that Ang II exacerbated TGF-induced gene expression of mesenchymal markers. This was not correlated with any changes in SMAD2 or ERK1/2 phosphorylation with co-stimulation of TGFβ and Ang II. However, superoxide production was significantly increased in HCAEC stimulated with Ang II but not TGFβ. Finally, the role of Ang II in microvesicle (MV)-mediated cardiomyocyte hypertrophy was investigated. MVs purified from neonatal rat cardiac fibroblasts were found to contain detectable Ang II and this was increased by stimulation of fibroblasts with Ang II. Treatment of cardiomyocytes with MVs derived from Ang II-stimulated fibroblasts induced cardiomyocyte hypertrophy which could be blocked by the AT1R antagonist losartan and an inhibitor of MV synthesis and release brefeldin A. Furthermore, Ang II was found to be present in MVs isolated from serum and plasma of Ang II-infused mice and SHRSP and WKY rats. Overall, the findings of this thesis demonstrate for the first time that the actions of Ang-(1-9) in cardiac pathology are dependent on its time of administration and that Ang-(1-9) can reverse Ang II-induced cardiac contractile dysfunction by acting as a positive inotrope. Furthermore, this thesis demonstrates evidence for an involvement of EndMT and MV signalling as novel pathways contributing to Ang II-induced cardiac fibrosis and hypertrophy, respectively. These findings provide incentive to further investigate the therapeutic potential of Ang-(1-9) in the treatment of cardiac contractile dysfunction in heart disease, establish the importance of novel pathways in Ang II-mediated cardiac remodelling and evaluate the significance of the presence of Ang II in plasma-derived MVs.