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Investigating the role of endothelin receptor subtypes in the response to vascular injury
Get PDFNeointimal hyperplasia, the proliferative growth of the innermost layer of the blood
vessel wall, is a key process in the response to vascular injury, underlying conditions
such as post-interventional restenosis and vein/arterial graft disease. One of the many
mediators implicated in this process is endothelin-1 (ET-1), a potent vasoconstrictor
with pro-inflammatory and pro-mitogenic actions, which acts through ETA and ETB
receptor subtypes. It is well established that ET-1 increases, and ETA blockade
reduces, neointima formation following vascular injury. The role of ETB is less clear
because these receptors mediate potentially beneficial actions in endothelial cells
(EC; such as nitric oxide production, and ET-1 clearance) but detrimental effects
elsewhere (such as vascular smooth muscle) and it has been recently reported that
non-cell-specific ETB deficiency is associated with increased neointimal lesion size
following injury. The work described in this thesis addressed the hypothesis that
endogenous ET-1 contributes to neointimal hyperplasia by activation of the ETA
receptor, and that this action is moderated by concurrent activation of the ETB
receptor expressed in EC.
The role of ET receptors in neointimal lesion development was assessed using two
models of femoral arterial injury in the mouse: (i) an established method of intraluminal
wire-injury, and (ii) adaptation of a model of ligation injury that induces
robust neointimal lesion formation without physical damage to the endothelium.
Lesion development was assessed using standard histological techniques and this
was augmented by development of quantitative optical projection tomography (OPT)
to allow three-dimensional analysis of lesions.
The role of ETA and ETB receptors in these models was addressed using suitable
pharmacological ET receptor antagonists. Following wire-injury, selective ETB
blockade (A192621; 30mg.kg-1.day-1; 35 days) increased lesion size and blood
pressure without significant altering lesion composition. In contrast, selective ETA
blockade (atrasentan; 10mg.kg-1.day-1; 35 days) reduced lesion size and blood
pressure. Combined ETA+ETB antagonism had no effect on lesion size, despite reducing blood pressure, and reducing collagen content of the lesions. In the ligation
model, neither ETA selective, ETB selective nor ETA+ETB blockade altered lesion
size as assessed by standard histology but analysis by OPT indicated that ETA
blockade, with or without concurrent ETB blockade, reduced lesion volume.
The influence of ETB receptors expressed by ECs on lesion formation was addressed
using EC-specific ETB knockout mice. Small vessel myography indicated that
endothelium-dependent relaxation was unaltered in femoral arteries from these mice.
In addition, no effect on lesion size or rate of development was observed in either
wire- or ligation-injury models of neointima formation (although subtle effects on
lesion and medial composition were apparent after intra-luminal injury).
These results indicate that ETB receptor activation can moderate the detrimental
actions of the ETA receptor on neointimal lesion progression, and that this role is
dependent on the mode of vascular injury. Furthermore, in this setting, this beneficial
action is not primarily mediated by ETB expressed by EC, suggesting that ETB in
other cell types can reduce lesion development through another, unidentified
mechanism. Therefore, while both ETA selective and non-selective ETA/B
antagonists are currently in clinical use, in conditions where similar arterial
remodelling processes occur, selective ETA receptor antagonists might be preferred
11β-Hydroxysteroid Dehydrogenase Type 1: a new therapeutic target post-myocardial infarction?
Get PDFGlucocorticoids can reduce infarct size when given immediately after myocardial infarction (MI) but are detrimental when administration is continued into the post-infarct healing phase. A number of experimental studies have shown that reduction of infarct expansion by enhancing blood supply to the infarct border reduces remodelling and improves heart function post-MI. Previous experiments from this laboratory have shown that mice unable to locally regenerate corticosterone due to deficiency in 11β-hydroxysteroid dehydrogenase type 1 (11HSD1) have an enhanced angiogenic response during myocardial infarct healing that is associated with improved cardiac function. We hypothesized that the enhanced angiogenic response in 11HSD1 knock out (-/-) mice would be preceded by augmented inflammation. Moreover this would be associated with improved cardiac function.
This thesis aimed firstly to establish that murine cardiac phenotype was not influenced by 11HSD1 deficiency. 11HSD1-/- and C57Bl6 control mice had comparable cardiac structure and function. 11HSD1 expression was localised to fibroblasts and vascular smooth muscle cells in the myocardium.
The second aim of this thesis was to characterise the healing response after MI in 11HSD1-/- mice compared to C57Bl6 mice. Neutrophil infiltration peaked 2 days after MI and was significantly enhanced in the 11HSD1-/- mice relative to C57Bl6 mice, despite comparable infarct size in both groups. This was followed by increased macrophage accumulation in the infarct border. Furthermore, in the 11HSD1-/- mice a greater proportion of macrophages were of the alternatively activated phenotype. Left ventricular expression of pro-angiogenic IL-8, but not VEGF, was increased. Cellular proliferation and vessel density at 7 days were greater in 11HSD1-/- compared to C57Bl6 hearts. This was associated with improved cardiac function 7 days post-MI.
The third aim of this thesis was to determine whether the enhancement in vessel density and cardiac function was maintained beyond the initial wound healing phase. 11HSD1-/- mice retained the increased vessel density compared to C57Bl6 mice and these vessels were smooth muscle coated suggesting vessel maturation. This was associated with sustained improvement in cardiac function and modification of the scar characteristics.
The final aim of this thesis was to establish whether the effect of the knock out could be recapitulated by administration of a small molecule inhibitor of 11HSD1 after MI. Oral administration of the 11HSD1 inhibitor had no effect on inflammation, angiogenesis and heart function as determined at 7 days post-MI relative to vehicle treated animals.
In conclusion, the data confirm the enhancement in vessel density and cardiac function in 11HSD1-/- mice and demonstrate that this was preceded by enhanced inflammation. This was not due to an underlying cardiac phenotype or modification of the infarct size. Increased infiltration of alternatively activated macrophages may have been the source of pro-angiogenic factor, IL-8, which was also increased at the time of angiogenesis. Importantly the enhanced vessel density was retained 4 weeks after MI, these vessels were mature suggesting longevity and the improvement in cardiac function was retained. While pharmacological inhibition did not recapitulate the effect of the knock out this may have been due to route of administration. The data provides compelling evidence that further development and use of small molecule inhibitors of 11HSD1 may be of benefit post-MI
Role of intra-cellular glucocorticoid regulation in vascular lesion development
Get PDFAtherosclerosis and post-angioplasty neointimal proliferation, which are leading causes of cardiovascular morbidity and mortality, develop as a result of chronic or acute vascular injury producing inflammatory and proliferative responses in the vessel wall.
Glucocorticoids, the stress hormones produced by the adrenal cortex, have anti-inflammatory and anti-proliferative characteristics and can also influence systemic cardiovascular risk factors. The systemic levels of these hormones are controlled by the hypothalamic pituitary adrenal axis. However, there is also a tissue-specific pre-receptor regulation of these hormones by the two isozymes of 11β-hydroxysteroid dehydrogenase (11β-HSD); type 1 regenerates active glucocorticoids within the cells and type 2 inactivates glucocorticoids. Whilst it has been shown that the inhibition of 11β-HSD1 has favourable effect on cardiovascular risk factors and the inhibition of 11β-HSD2 results in hypertension; the effect of these enzymes on vascular lesion development is not known. The work described in this thesis tested the hypothesis that 11β-HSD1 inhibition reduces vascular lesion development due to improvement in cardiovascular risk factors, whereas 11β-HSD2 inhibition leads to adverse vascular remodelling.
Apolipoprotein-E deficient (ApoE-/-) mice fed on western diet were used to study atherosclerosis, whereas neointimal proliferation was investigated using a well-established mouse model of wire-angioplasty. Vascular lesions were assessed using novel imaging and standard histological techniques.
11β-HSD1 inhibition reduced the size of atherosclerotic lesions and improved markers of plaque stability with a reduction in lipid content and increase in collagen content of the plaques. This was associated with a reduction in weight gain and blood pressure but without any effect on lipid profile. 11β-HSD1 inhibition did not produce any significant effect on neointimal proliferation in C57Bl/6J mice. However in ApoE-/- mice, 11β-HSD1 inhibition reduced neointimal proliferation with corresponding increase in size of patent lumen and with an associated reduction in macrophage content of neointimal lesions. 11β-HSD2 deletion produced an outward remodelling in un-injured vessels but there was no effect on neointimal proliferation after wire-angioplasty. Administration of a selective mineralocorticoid antagonist, eplerenone, reduced neointimal lesions
significantly but to a similar degree in both C57Bl/6J and 11β-HSD2-/- mice, associated with a significant reduction in macrophage content of lesions but without any effect on blood pressure.
Data in this thesis highlight the potential therapeutic application of 11β-HSD1 inhibition in reducing the size and vulnerability of atherosclerotic plaques and also reduction in neointimal proliferation (and hence post-angioplasty restenosis) in high risk patients with „metabolic syndrome‟ phenotype. The results also indicate that 11β-HSD2 has a limited, if any, role to play in the development of neointimal lesions
Role of murine 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) in the metabolism of 7-oxysterols
Get PDF7-Oxysterols constitute the major component (40%) of oxidized low-density
lipoprotein (oxLDL). They arise in the body via auto-oxidation of cholesterol and are
known to induce endothelial dysfunction, oxidative stress and apoptosis in the
vascular wall, prior to development of atherosclerosis. A novel pathway has been
described for hepatic inter-conversion of 7-ketocholesterol (7-KC) and 7β -hydroxycholesterol
(7β OHC) by the enzyme 11β-hydroxysteroid dehydrogenase type-1
(11β HSD1), better known for metabolizing glucocorticoids. Inhibition of 11βHSD1
is atheroprotective and the potential underlying mechanism for this may involve
altered metabolism and actions of glucocorticoids. However, alterations in the
metabolism of 7-oxysterols may also play an important role in this atheroprotective
effect.
The work described here addresses the hypotheses that (i) 7-oxysterols are substrates
for murine 11βHSD1; (ii) inhibition of 11β HSD1 may abolish cellular metabolism of
7-oxysterols; (iii) this route of metabolism may modulate the actions of 7-oxysterols
and glucocorticoids on murine vascular physiology.
Murine 11β HSD1 inter-converted 7-oxysterols (Km=327.6±98ìM,
Vmax=0.01±0.001pmol/ìg/min) but the regulation of reaction direction is different
from that for glucocorticoids. Predominant dehydrogenation of 7β OHC to 7-KC was
quantified in several models (recombinant protein, cultured cells stably transfected
with 11β HSD1), in which predominant reduction of glucocorticoids was measured.
Furthermore, in murine hepatic microsomes, dehydrogenation of 7β OHC occurred
exclusively. In aortic rings in culture, however, both reduction and dehydrogenation
of 7-oxysterols were evident. 7-Oxysterols and glucocorticoid substrates competed
for metabolism by 11β HSD1, with 7β OHC inhibiting dehydrogenation of
glucocorticoids (Ki=908±53nM).
The circulating concentrations of 7-oxysterols in the plasma of C57Bl6 and
11β HSD1-/- mice were in the ìM range (0.02 – 0.13ìM). The disruption of
11β HSD1 has resulted in increased ratios of 7-KC and 7β OHC over total plasma
cholesterol levels (*p<0.05). This finding suggested that 11β HSD1 is involved in
metabolizing and determining the plasma levels of 7-KC and 7β OHC.
To assess the consequences of these alterations for vascular function, studies were
undertaken in aortic rings. Prolonged incubation with 7-oxysterols (20-25 ìM)
showed a tendency to attenuate noradrenaline-mediated contractions of C57Bl6
aortae, but had no effect on contractions in response to 5-hydroxytryptamine or KCl.
Similarly, endothelium-dependent and -independent relaxations of murine aortae
were unaltered after exposure to 7-oxysterols.
Thus in the mouse, 11β HSD1 may influence the balance of circulating and cellular
7-oxysterols which may have consequential effects on glucocorticoid action.
Although this work suggests that concentrations present in murine tissues are
unlikely to cause vascular dysfunction, they may influence further cellular events as
yet undescribed. Under pathological conditions where high concentrations of
7-oxysterols occur, 11β HSD1 may influence the extracellular-transport and delivery
of 7-KC and 7β OHC to the plaque. This work therefore proposes that inhibition of
metabolism of 7-oxysterols by 11β HSD1 inhibitors, may contribute to the
atheroprotective effects of these drugs
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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