Audrey Dooley

Current developments and future prospects for heart valve replacement therapy.

Valve replacement is the most common surgical treatment in patients with advanced valvular heart disease. Mechanical and bio-prostheses have been the traditional heart valve replacements in these patients. However, currently the heart valves for replacement therapy are imperfect and subject patients to one or more ongoing risks, including thrombosis, limited durability, and need for re-operations due to the lack of growth in pediatric populations. Furthermore, they require an open heart surgery, which is risky for elderly and young children who are too weak or ill to undergo major surgery. This article reviews the current state of the art of heart valve replacements in light of their potential clinical applications. In recent years polymeric materials have been widely studied as potential prosthetic heart valve material being designed to overcome the clinical problems associated with both mechanical and bio-prosthetic valves. The review also addresses the advances in polymer materials, tissue engineering approaches, and the development of percutaneous valve replacement technology and discusses the future prospects in these fields.

Does Periadventitial Fat-Derived Nitric Oxide Play a Role in Improved Saphenous Vein Graft Patency in Patients Undergoing Coronary Artery Bypass Surgery?

Background/Aims: The saphenous vein is commonly used for coronary artery bypass surgery but its patency is poor. Vascular damage occurs during conventional surgery. However, patency improves when the graft is harvested with minimal surgical trauma, partly due to preservation of vascular endothelial nitric oxide synthase (eNOS) and tissue sources of nitric oxide (NO), a factor possessing both dilatory and anti-proliferative properties. Apart from these grafts exhibiting an intact luminal endothelium they are harvested complete with a surrounding cushion of tissue, much of which is fat. Methods: Immunostaining for eNOS was performed on vein graft sections and reverse-transcriptase polymerase chain reaction and Western blotting were used to identify eNOS mRNA and protein. NO synthase activity was measured using the citrulline assay. Results: Immunohistochemistry identified eNOS staining of vein graft segments, including dense staining of the cushion of perivascular fat and associated structures surrounding the vein. eNOS protein was confirmed in both the vein and surrounding fat by Western blot analysis. Using the citrulline assay, the perivascular fat and underlying vein possessed comparable NO synthase activity. Conclusions: Our observations suggest that perivascular fat-derived NO plays a beneficial role in saphenous veins harvested atraumatically and used as grafts in patients undergoing coronary artery bypass surgery.

Serotonin promotes the survival of cortical glutamatergic neurons in vitro

The appearance of 5-hydroxytryptamine (serotonin; 5-HT) in the cerebral cortex coincides with developmental events such as cell proliferation, survival, and differentiation. We tested the hypothesis that 5-HT plays a role in these events by examining rat cortical progenitor cells in vitro. Using bromodeoxyuridine incorporation we found that 5-HT did not affect the proliferation of these cells, but a cell survival assay indicated that it promoted their survival. The observed survival effect was mimicked by the 5-HT2a/2c receptor agonist alpha-methyl-5-HT and blocked by the 5-HT2a receptor antagonist cinanserin. Consistent with increased survival was the finding, using the terminal transferase nick end labeling method, of reduced cell death in cultures exposed to 5-HT. Immunohistochemical analysis with cell-specific markers revealed that the effect of 5-HT was directed specifically to the glutamate-containing neuronal population and not to any other cortical cell types. These results indicate that 5-HT does not exert its effects on dividing neuroepithelial cells in the developing cortex, but rather on postmitotic neurons.

Modulation of collagen type I, fibronectin and dermal fibroblast function and activity, in systemic sclerosis by the antioxidant epigallocatechin-3-gallate

OBJECTIVES SSc is characterized by the overproduction of extracellular matrix (ECM) proteins, such as collagen and fibronectin, by activated fibroblasts, as well as oxidative stress. This study investigates the anti-fibrotic potential of the antioxidant epigallocatechin-3-gallate (EGCG) on activated dermal fibroblasts from SSc patients. METHODS Dermal fibroblasts from a cell line (AG), healthy individuals (CON) and SSc patients were treated with EGCG, TGF-β, PDGF-BB or other antioxidants [antioxidants superoxide dismutase (SOD), catalase, N-acetyl-L-cysteine (NAC) and diphenyleneiodonium (DPI)]. Collagen type I, fibronectin, connective tissue growth factor (CTGF), α-smooth muscle actin and mitogen-activated protein (MAP) kinases were measured by ELISA and western blot. Fibroblast contractile forces were measured by collagen gel contraction. Reactive oxygen species (ROS) were assessed by dichlorofluorescein assay and nuclear factor κ beta (NF-κB) activity by DNA binding assay. RESULTS EGCG (1-100 µM) dose-dependently decreased collagen type I secretion in culture medium after 24 h in AG fibroblasts. Collagen type I protein expression in cell lysates was also significantly reduced by 40% in EGCG-treated cells (40 µM). Furthermore, EGCG also down-regulated TGF-β-induced collagen type I, fibronectin and CTGF. Similarly, in CON fibroblasts EGCG decreased basal and stimulated collagen type I, fibronectin and CTGF after 24 h, while in SSc the effects of the antioxidant were apparent after 48 h. Fibroblast-mediated contraction of collagen gels was inhibited by EGCG as early as 1 h in AG fibroblasts, and in the CON and SSc fibroblasts. Additionally, EGCG also inhibited TGF-β-stimulated gel contraction similar to other antioxidants DPI and NAC, but not SOD or catalase. EGCG suppressed TGF-β-induced ROS production in all fibroblasts. Furthermore, EGCG inhibited TGF-β or PDGF-BB-induced phospho-extracellular signal-regulated kinase (ERK)1/2 MAP kinase and NF-κB activity in SSc fibroblasts. CONCLUSION The results suggest that the antioxidant, EGCG, can reduce ECM production, the fibrotic marker CTGF and inhibit contraction of dermal fibroblasts from SSc patients. Furthermore, EGCG was able to suppress intracellular ROS, ERK1/2 kinase signalling and NF-κB activity. Taken together, EGCG may be a possible candidate for therapeutic treatment aimed at reducing both oxidant stress and the fibrotic effects associated with SSc.

Endothelial Injury in a Transforming Growth Factor β–Dependent Mouse Model of Scleroderma Induces Pulmonary Arterial Hypertension

OBJECTIVE To delineate the constitutive pulmonary vascular phenotype of the TβRIIΔk-fib mouse model of scleroderma, and to selectively induce pulmonary endothelial cell injury using vascular endothelial growth factor (VEGF) inhibition to develop a model with features characteristic of pulmonary arterial hypertension (PAH). METHODS The TβRIIΔk-fib mouse strain expresses a kinase-deficient transforming growth factor β (TGFβ) receptor type II driven by a fibroblast-specific promoter, leading to ligand-dependent up-regulation of TGFβ signaling, and replicates key fibrotic features of scleroderma. Structural, biochemical, and functional assessments of pulmonary vessels, including in vivo hemodynamic studies, were performed before and following VEGF inhibition, which induced pulmonary endothelial cell apoptosis. These assessments included biochemical analysis of the TGFβ and VEGF signaling axes in tissue sections and explanted smooth muscle cells. RESULTS In the TβRIIΔk-fib mouse strain, a constitutive pulmonary vasculopathy with medial thickening, a perivascular proliferating chronic inflammatory cell infiltrate, and mildly elevated pulmonary artery pressure resembled the well-described chronic hypoxia model of pulmonary hypertension. Following administration of SU5416, the pulmonary vascular phenotype was more florid, with pulmonary arteriolar luminal obliteration by apoptosis-resistant proliferating endothelial cells. These changes resulted in right ventricular hypertrophy, confirming hemodynamically significant PAH. Altered expression of TGFβ and VEGF ligand and receptor was consistent with a scleroderma phenotype. CONCLUSION In this study, we replicated key features of systemic sclerosis-related PAH in a mouse model. Our results suggest that pulmonary endothelial cell injury in a genetically susceptible mouse strain triggers this complication and support the underlying role of functional interplay between TGFβ and VEGF, which provides insight into the pathogenesis of this disease.

Systemic vasculopathy with altered vasoreactivity in a transgenic mouse model of scleroderma

IntroductionVasculopathy, including altered vasoreactivity and abnormal large vessel biomechanics, is a hallmark of systemic sclerosis (SSc). However, the pathogenic link with other aspects of the disease is less clear. To assess the potential role of transforming growth factor beta (TGF-β) overactivity in driving these cardiovascular abnormalities, we studied a novel transgenic mouse model characterized by ligand-dependent activation of TGF-β signaling in fibroblasts.MethodsThe transgenic mouse strain Tβ RIIΔk-fib is characterized by balanced ligand-dependent upregulation of TGF-β signaling. Aortic and cardiac tissues were examined with histologic, biochemical, and isolated organ bath studies. Vascular and perivascular architecture was examined by hematoxylin and eosin (H&E) and special stains including immunostaining for TGF-β1 and phospho-Smad2/3 (pSmad2/3). Confirmatory aortic smooth muscle cell proliferation, phenotype, and functional assays, including signaling responses to exogenous TGF-β and endothelin-1, were performed. Aortic ring contractile responses to direct and receptor-mediated stimulation were assessed.ResultsAortic ring contractility and relaxation were diminished compared with wild-type controls, and this was associated with aortic adventitial fibrosis confirmed histologically and with Sircol assay. TGF-β1 and pSmad 2/3 expression was increased in the adventitia and smooth muscle layer of the aorta. Aortic smooth muscle cells from transgenic animals showed significant upregulation of TGF-β- responsive genes important for cytoskeletal function, such as transgelin and smoothelin, which were then resistant to further stimulation with exogenous TGF-β1. These cells promoted significantly more contraction of free floating type I collagen lattices when compared with the wild-type, but were again resistant to exogenous TGF-β1 stimulation. Aortic ring responses to receptor-mediated contraction were reduced in the transgenic animals. Specifically, bosentan reduced endothelin-mediated contraction in wild-type animals, but had no effect in transgenic animals, and endothelin axis gene expression was altered in transgenic animals. Transgenic mice developed cardiac fibrosis.ConclusionsThe histologic, biochemical, and functional phenotype of this transgenic mouse model of scleroderma offers insight into the altered biomechanical properties previously reported for large elastic arteries in human SSc and suggests a role for perturbed TGF-β and endothelin activity in this process.

Modulation of fibrosis in systemic sclerosis by nitric oxide and antioxidants.

SYSTEMIC SCLEROSIS (SCLERODERMA: SSc) is a multisystem, connective tissue disease of unknown aetiology characterized by vascular dysfunction, autoimmunity, and enhanced fibroblast activity resulting in fibrosis of the skin, heart, and lungs, and ultimately internal organ failure, and death. One of the most important and early modulators of disease activity is thought to be oxidative stress. Evidence suggests that the free radical nitric oxide (NO), a key mediator of oxidative stress, can profoundly influence the early microvasculopathy, and possibly the ensuing fibrogenic response. Animal models and human studies have also identified dietary antioxidants, such as epigallocatechin-3-gallate (EGCG), to function as a protective system against oxidative stress and fibrosis. Hence, targeting EGCG may prove a possible candidate for therapeutic treatment aimed at reducing both oxidant stress and the fibrotic effects associated with SSc.

Gut fibrosis with altered colonic contractility in a mouse model of scleroderma

OBJECTIVE Gastrointestinal involvement occurs in up to 90% of patients with SSc. Animal models of SSc mimic some of the pathophysiological disease processes of SSc. The transgenic (TG) mouse strain TβRIIΔk-fib is characterized by ligand-dependent up-regulation of TGF-β signalling and has been shown to develop skin fibrosis, lung fibrosis and diminished aortic ring contractility and adventitial fibrosis. We investigated if similar changes are observed in the gut tissue in this mouse model. METHODS Colonic tissue was examined using histology and immunohistochemistry analyses. Tissue architecture was examined by haematoxylin and eosin (H&E), picrosirius red and immunohistochemical markers for α-smooth muscle actin (α-SMA), phospho-Smad 2/3 (pSmad2/3), Ki-67, protein gene product 9.5 and S-100. Fibrosis was quantified using the NIS Elements BR 2.30 system and by Sircol assay. Colonic strip contractile responses to potassium chloride (KCl) and carbachol were assessed in isolated organ baths. Confirmatory gut fibroblast and intestinal tissue biochemical assays, including cellular signalling mechanisms, were performed. RESULTS H&E staining and staining for α-SMA, Ki-67, pSmad2/3 or neural tissue staining showed no differences between TG and wild-type (WT) mice gut tissue. There was increased collagen deposition in the gut of TG mice. Quantitative PCR results of TG gut fibroblasts showed evidence of up-regulated collagen and CTGF transcription, and non-canonical TGF-β signalling pathways were also up-regulated. The organ bath studies showed diminished colonic strip contractility in TG mice compared with WT control mice to KCl and carbachol. CONCLUSION We have shown that this TG mouse model, previously shown to develop skin and lung, develops colonic fibrosis with associated effects on colonic tissue contractility. This may offer further insight in pathological processes leading to the development of gut fibrosis.

The Vasa Vasorum and Associated Endothelial Nitric Oxide Synthase is More Important for Saphenous Vein Than Arterial Bypass Grafts

No-touch (NT) saphenous vein (SV) grafts are superior to SVs harvested by the conventional technique (CT), with a patency comparable with the internal thoracic artery (ITA). Preservation of the vasa vasorum is implicated in the success of NT harvesting. We compared the vasa vasorum and endothelial nitric oxide synthase (eNOS) in NT SV with ITA and radial artery (RA) grafts. Skeletonized SV (SSV) was also analyzed. The NT SV had a higher number and larger vasa vasorum compared with ITA (P = .0001) and RA (P = .0004) that correlated with eNOS protein. Activity of eNOS in SSV grafts was significantly lower than NT SV grafts (P = 004). Since a high proportion of the vasa vasorum are removed in SSV using the CT, we suggest that preservation of the vasa vasorum and eNOS-derived NO contributes to the high patency for NT as compared with SSV grafts.

Pulmonary endothelial injury in the context of perturbed transforming growth factor β signalling as a unique model of pulmonary hypertension in scleroderma

Abstract Background The development of pulmonary arterial hypertension in scleroderma remains an important contributor to mortality in this condition, despite substantial improvements in outcomes due to modern therapeutic strategies. No animal models of scleroderma develop this important complication. We describe the constitutive vascular phenotype of a mouse model of scleroderma and show that pulmonary endothelial injury replicates the pathological changes of pulmonary arterial hypertension seen in human disease. Methods The TβRIIΔk-fib mouse strain expresses a kinase-deficient type II transforming growth factor β (TGFβ) receptor driven by a fibroblast-specific promoter leading to ligand-dependent upregulation of TGFβ signalling; this mouse strain replicates key fibrotic features of scleroderma. We did structural, biochemical, and functional assessments of pulmonary and systemic vessels, including in-vivo haemodynamic studies, before and after vascular endothelial growth factor receptor (VEGFR) inhibition with SU5416, which induced pulmonary endothelial cell apoptosis. These assessments included biochemical analysis of the TGFβ, endothelin, and VEGF signalling axes in vivo; tissue sections; and explanted pulmonary arterial smooth muscle cells. Findings In the TβRIIΔk-fib mouse strain, a constitutive pulmonary vasculopathy with medial thickening, a perivascular proliferating chronic inflammatory cell infiltrate, and mildly raised pulmonary artery pressures resemble the well-described chronic hypoxia model of pulmonary hypertension. After administration of SU5416, the pulmonary vascular phenotype was more florid, with pulmonary arteriolar luminal obliteration by apoptosis-resistant proliferating endothelial cells; the result was right ventricular hypertrophy confirming haemodynamically significant pulmonary arterial hypertension. Altered TGFβ, endothelin, and ligand and receptor expression of VEGF were consistent with a scleroderma phenotype. Interpretation This study replicates key features of scleroderma-associated pulmonary arterial hypertension in a mouse model. Our results suggest that pulmonary endothelial cell injury in a genetically susceptible mouse strain triggers this complication and support functional interplay between TGFβ, endothelin, and VEGF that provides insight into pathogenesis. Funding Arthritis Research UK.