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7.1.1 Study rationale

Obesity and its vascular complications such as hypertension and type-II diabetes, are major risk factors in CVD; the leading cause of death worldwide (British Heart Foundation, 2017; World Health Organisation, 2017). It is estimated that 1.9 billion adults and 41 million children worldwide are obese, and these numbers are growing. These figures present a considerable burden to society, and highlight the need for novel and effective treatments of the complications of obesity. PVAT surrounds the majority of blood vessels, and a number of studies have demonstrated that PVAT releases vasoactive factors which contribute to the regulation of vascular resistance (Greenstein et al., 2009; Galvez-Prieto et al., 2012; de Boer et al., 2016; Torok et al., 2016). These factors may be important in regulating blood pressure, and blood glucose, and the loss of function which occurs in obesity, may be contributing to the development of hypertension and type-II diabetes, however the mechanisms underlying these effects remain unclear. Sympathetic nerves are present within PVAT (Slavin & Ballard, 1978; Rebuffe-Scrive, 1991); however evidence of direct communication between nerves and adipocytes is limited. It is well known that autonomic dysfunction occurs in obesity (Smith & Minson, 2012; Manolis et al., 2014), and sympathetic over-activity may be causing desensitisation of the PVAT mechanism. Accordingly, we have investigated the function of sympathetic nerves in healthy PVAT, and how this changes in obesity. In addition, healthy sympathetic nervous stimulation by exercise, has been shown to have positive effects on inflammation in obesity in skeletal muscle and abdominal fat (Bradley et al., 2008; Kawanishi et al., 2010; Samaan et al., 2014), therefore we have studied the potential for exercise in restoring PVAT function in obesity.

The aims of this thesis were as follows:

1. Elucidate the mechanism by which sympathetic nervous discharge leads to a PVAT anti-contractile effect.

2. Identify adrenoceptors, transporters, and intramural nerves within PVAT.

3. Compare healthy PVAT with the PVAT of animals with pathological sympathetic nervous system activity induced by obesity.

4. Investigate the effects of healthy sympathetic nervous stimulation on dysfunctional PVAT.

5. Demonstrate the role of PVAT in modulating vascular tone in vivo.

7.1.2 Aims 1 & 2

Using immunohistochemistry we identified sympathetic nerves, β3-adrenoceptors and OCT3,

within mouse mesenteric PVAT, and confirmed functional roles for each of these components in the PVAT anti-contractile effect using various pharmacological tools in the wire myograph. We

156 found that electrical activation of sympathetic nerves within PVAT stimulates an anti-contractile effect, which could be abolished by pharmacologically denervating the PVAT. The data presented in this thesis provide convincing evidence that the roles of PVAT are two-fold. First, the sympathetically derived NA activates adipocyte β3-adrenoceptors, triggering the release of

an anti-contractile factor from adipocytes. Second, PVAT acts as a “sponge” for NA, by transporting NA into the adipocytes via OCT3, thereby preventing NA from reaching the blood vessel and eliciting contraction. It is widely accepted that sympathetic nerve derived catecholamines activate β3-adrenoceptors to trigger lipolysis (Rebuffe-Scrive, 1991; Robidoux et

al., 2006), however this is the first study to confirm that β3-adrenoceptors contribute to the PVAT

anti-contractile effect, and therefore modulate vascular resistance. Previous studies have highlighted the role of OCT3 in NA transport into adipocytes, where it is broken down by MAO-A (Pizzinat et al., 1999; Ayala-Lopez et al., 2015); however the physiological importance of this transport is unknown. Again for the first time, we have demonstrated that this role of OCT3 in transporting NA into adipocytes is vital in PVAT’s contribution to vascular tone. This neurally evoked PVAT anti-contractile effect likely contributes to the modulation of blood pressure, and delivery of nutrients i.e. glucose into skeletal muscles.

Consistent with previous studies of the NA induced anti-contractile effect (Fesus et al., 2007; Greenstein et al., 2009; Lynch et al., 2013), the results of this study suggest that adiponectin is the anti-contractile factor released upon β3-adrenoceptor activation. We have also found that

the anti-contractile effect is NOS-dependent. It is likely that NO has a number of roles in the PVAT anti-contractile effect including increasing adiponectin production, modulating OCT3 activity or expression, and acting as an anti-contractile factor (Haas et al., 2009; Schneider et al., 2011). Similar to adiponectin, these results are consistent with other studies of the NA induced anti-contractile effect (Greenstein et al., 2009; Bussey et al., 2016). Moreover, we have confirmed the expression of eNOS in mouse mesenteric PVAT, supporting previous suggestions that eNOS is the isoform most likely to be important in PVAT (Dashwood et al., 2007; Bussey et al., 2016). However, we have not investigated the expression of nNOS and inducible NOS (iNOS), therefore further study is required to dissect out the exact roles of NO, and examine the expression of the different NOS isoforms.

7.1.3 Aim 3

We developed a high fat feeding mouse model of obesity, which exhibited the metabolic syndrome, namely hypertension, abdominal obesity, and type-II diabetes. In the obese PVAT, we found that the expression of β3-adrenoceptors and OCT3 may be reduced, and the PVAT

anti-contractile function was lost. Whilst a reduction in β3-adrenoceptors in obesity has already

been described, we are the first to suggest a reduction in OCT3 expression (Collins et al., 1999). These mice also exhibited a dysfunctional sympathetic nervous system, which may suggest that that pathological over-activity of the sympathetic nervous system that occurs in obesity (Smith & Minson, 2012; Manolis et al., 2014), may cause desensitisation of β3-

157 increase in the expression of TNFα, a marker of inflammation, in obese PVAT. Sympathetic nerves play a role in recruiting inflammatory mediators (Levick et al., 2010); therefore sympathetic dysfunction may be contributing to increased inflammation of PVAT in obesity. Inflammation will damage the PVAT environment, therefore contributing to a loss of function. Loss of the PVAT anti-contractile mechanism may be contributing to the development of hypertension and type-II diabetes in obese patients; therefore it was vital that we investigate PVAT dysfunction in obesity, in order to identify new targets for intervention in the vascular complications of obesity.

We found that pharmacological activation of β3-adrenoceptors had no effect on rescuing the

loss of PVAT function. Moreover, we found that exogenous application of adiponectin no longer exerted vasodilation in obese vessels, despite no change in AdipoR1 expression. These results eliminate targeting β3-adrenoceptor activation, and application of adiponectin as potential

therapeutics for targeting vascular disease in obesity. However, we did find that non-specific activation of NOS rescued PVAT function in obesity, and expression of eNOS may be unchanged; indicating a possible target for the treatment of obesity-related hypertension and type-II diabetes.

7.1.4 Aim 4

As consistent with previous exercise studies, we have shown that exercise improves blood pressure, blood glucose, and plasma insulin in obesity (Reisin & Jack, 2009; Riddell et al., 2013; Abdelaal & Mohamad, 2015). In our study, we have found that these improvements were independent of weight-loss. The PVAT environment was studied immunohistochemically, and we found that exercise may have induced improvements in inflammation, and expression of β3-

adrenoceptors and OCT3. Moreover, in the wire myograph we found that the PVAT anti- contractile effect, and its normal mechanism, had been restored. This is the first study to describe the beneficial effects of exercise on the PVAT environment and function. This reversal of PVAT dysfunction in obesity may be contributing to the improvements in vascular function in obesity, highlighting the potential benefits of exercise as a first line of treatment of the vascular complications of obesity.

7.1.5 Aim 5

The results of this study describe for the first time a PVAT anti-contractile effect in vivo. We found that PVAT, which has been well studied in vitro (Greenstein et al., 2009; Galvez-Prieto et al., 2012; de Boer et al., 2016; Torok et al., 2016), elicits a paracrine effect on the vasculature in vivo. By repeating some of our control in vitro experiments in the in vivo preparation, we surmise that a similar sympathetically-mediated PVAT anti-contractile mechanism exists in vivo, which when active may have direct effects on the regulation of blood pressure, and nutrient delivery. The results of this study also support the translation of our obese and exercise studies in vivo. These findings highlight the physiological relevance of PVAT studies, and stress the importance of understanding PVAT dysfunction in obesity, so that novel and effective therapeutics may be

158 developed.

7.1.6 Key findings

The main findings of this study are as follows: 1) Sympathetic nerves elicit an anti-contractile effect dependent on adipocyte β3-adrenoceptors and OCT3. 2) Adiponectin is the anti-

contractile factor released upon β3-adrenoceptor activation. 3) Sympathetic dysfunction in diet

induced obesity may lead to downregulation of β3-adrenoceptors and OCT3, leading to a loss of

function. 4) Activation of NOS in obese PVAT can restore loss of function. 5) Exercise may restore the PVAT anti-contractile effect by reducing inflammation, and upregulation of β3-

adrenoceptors and OCT3. 6) The PVAT anti-contractile effect is present in vivo.

7.1.7 Overall conclusion

Sympathetic nerves within PVAT elicit an anti-contractile effect, which may be vital in modulation of blood pressure and nutrient delivery. Therefore, the loss of this neurally-mediated PVAT function in obesity may contribute to the development of hypertension and type-II diabetes in obese patients. However, healthy sympathetic nervous stimulation by exercise restores PVAT function, and protects against the vascular complications of obesity, highlighting the importance of exercise as the first line of treatment in obesity.

7.2

Limitations

7.2.1 The clinical relevance of animal models

Animal models are commonly used to study obesity, and there are a number of advantages to using animal tissue. Human tissue would provide a better insight into human physiology, however tissue samples are limited, as well as access to physiologically relevant vessels. From humans, subcutaneous vessels can be biopsied, however it is mesenteric resistance vessels which play an important role in regulating blood pressure (Marieb & Hoehn, 2010); which are easily isolated from animal models. Moreover, it is easy to control environmental factors such as dietary intake in animals; reducing variability. Additionally, whilst diet and exercise are one the first lines of treatment of obesity (Kotsis et al., 2010), humans are less likely to adhere strictly to a diet and exercise regime, whereas an exercise protocol in animals can be forced, and as already mentioned, dietary intake can be monitored. Whilst it may be advantageous to use animals, care must be taken when interpreting results, as there are likely to be species differences (Shanks et al., 2009). However, the findings of this study are comparable to previous studies of the PVAT anti-contractile effect in humans, strengthening the translation of our results into humans (Greenstein et al., 2009; Aghamohammadzadeh et al., 2013).

In humans, lifestyle is the most common cause of obesity (National Health service, 2017a; World Heart Federation, 2017); therefore a high-fat feeding animal model of obesity is the most physiologically relevant. In some humans, obesity resistance can occur (Ding et al., 2015), and this can also occur in rodents (Chang et al., 1990). Therefore before commencing the HFD study, we decided to exclude any HFD mice with less than a 10% increase in body weight as

159 compared to controls, as a 10% increase is considered moderately obese in rodents (Hariri & Thibault, 2010). Another factor to consider is mouse behaviour; we observed that in each cage of five mice there is a hierarchy which is established by fighting, and generally there is an alpha mouse, and usually one mouse at the bottom of hierarchy. Whilst this occurs in all cages, it was particularly evident in HFD cages, where the alpha mouse would become severely obese, and the mouse at the bottom of the hierarchy would gain very little weight. Our 10% exclusion criteria allowed us to exclude these mice.

There are known differences in vascular responsiveness and adipose distribution between human males and females (Demerath et al., 2007; Fuente-Martin et al., 2013); however during the course of this study, only male mice were used. This was to eliminate hormonal variations that occur with the oestrus cycle in females. Previous studies in PVAT also only used males, therefore it is not known if the PVAT mechanism and function varies with gender, and requires further study.

7.2.2 Expression studies

Whilst immunohistochemistry is useful in illustrating the presence of proteins, analysis is qualitative and not quantitative, and can be subjective. Therefore, care must be taken when interpreting results. However, a good correlation between immunohistochemistry and other quantitative protein detection techniques such as ELISAs and western blotting has been shown (Becker, 1993; True, 1988), therefore immunohistochemistry is at least a good indicator of changes in protein expression. To definitively determine protein changes, and measure the amount of protein present, the immunohistochemical findings of this study would be complemented by western blotting. However, previous studies in our laboratory have found that protein content of PVAT is very low, which makes western blotting difficult, and results difficult to replicate. A large amount of PVAT is needed in order to detect our proteins of interest, and PVAT from mice would need to be pooled. From control mice, where there is substantially less mesenteric PVAT than in the HFD mice, we estimate that PVAT from around ten mice would need to be pooled; therefore it was decided not to employ western blotting to study the expression of our proteins of interest at this time; however PVAT samples were collected and stored for potential future study. In addition, changes in protein expression do not indicate changes in activity. Activity assays could be used to examine changes in activity in disease states.

7.2.3 Interpreting wire myography results

As already discussed in chapter 6 section 6.1, wire myography has been criticised for the study of PVAT, as this technique uses open ended vessels which allows components released from the PVAT to interact with the intraluminal side of the vessel, which would not occur in vivo

(Gollasch, 2012). We have addressed this limitation somewhat by repeating some of our control experiments performed in the myograph, in our in vivo setup. However, the obese and exercise studies of PVAT have not yet been studied in vivo; therefore careful interpretation of our results

160 is needed. Since both the in vitro and in vivo control experiments yielded similar results, this does strengthen translation of our obese and exercise results into in vivo conditions.

Additionally, using isolated arteries ex vivo eliminates the influence of factors circulating in the bloodstream, which may play a role in vivo. And whilst pharmacologically, we can manipulate β3-adrenoceptors, OCT3, NOS, and adiponectin individually to examine their functional

significance, there may be other receptors and pathways that are still contributing and affecting results. To further dissect out the molecular mechanisms, and examine the interactions between adipocytes and VSMCs, cell culture could be used. Using this technique, it would also be possible to manipulate protein expression, and mimic over- and under-expression of proteins that may be occurring in disease states. However, again care must be taken as VSMCs lose some of their contractile properties in culture, and therefore cultured VSMCs are thought to be better models of diseased states than health (Proudfoot & Shanahan, 2012).