Pathological Society iBSc Grant Report
Jennifer Kwan
The impact of menopause and oestrogen replacement therapy on cardioprotection in
remote ischaemic preconditioning
With the time-constraints of the laboratory work behind me, and my thesis finally handed in, I can
breathe a huge sigh of short-lived relief. Unlike what I’d envisioned the iBSc to be, I’ve ended up
working harder than I had ever had. Perhaps it was the culmination of the gruelling day-to-day
experiments, or the tangible grasp of a freshly-printed thesis in hand, but I feel strangely proud of
my accomplishments over the past year. These feelings stem from a sense of self-worth and
acknowledgement of personal change, for which I attribute to the iBSc experience and am most
grateful for the opportunity enabled by your Pathological Society grant.
Ironically I did get a break away from medicine in that I have worked under a very different academic
environment. The pressures in research are unlike ward work and allow more time to indulge in
scientific curiosity. The monotony and inevitable frustration of re-doing experiments, particularly
when they failed, was spurred on by a child-like enthusiasm to try out new things and discover new
secrets. The months of stagnation at the beginning of the year when nothing seemed to work was
paradoxically an enriching time because I built up resilience, a work ethic, and scientific questioning
in pursuit of a novel model of isolated cardiomyocyte ischaemia/reperfusion injury.
There were times of mounting stress and pressure of deadlines looming together. This revolved
around grant applications, abstract writing, poster submissions and presentations. I did wonder
whether academic medicine was a suitable career choice, and whether I would never venture into a
laboratory again. I felt the futility of examining a specialised niche of science if I wasn’t going to
change the practice of medicine and patients’ lives. But, I am no genius.
At the very least, I have managed to put theory to test, answered a clinical question, and
communicated data with others at conferences and meetings. I have been able to present at the
National Student Association for Medical Research Conference 2014, and the British Society for
Cardiovascular Research Spring Meeting 2014, then there is the upcoming Physiology 2014 Rob
Clarke Awards in July – all of which I have acknowledged the contribution of your Society. These
sessions have provided me with a platform to practise communicating my findings with respected
individuals from other fields, and overcome my feeling of inferiority as a lowly BSc student. I have
been inspired by the work of other students, as well as by brief encounters with well-established
researchers. My moment of epiphany was when I suddenly realised, “Oh, I understood that – and I
can go back and do this…and…that…” I felt I had collaborated and networked with scientists, and
was finally a part of the scientific community.
I realised the bigger picture of basic science is that it can lead to translational medicine which leads
to clinical practice – there is no need for a Nobel prize to recognise results, though it would be nice!
The foundation of my project was based on the mounting evidence for endothelial dysfunction, as
measured by endothelium-dependent vasodilation, in post-menopausal women (1-3). Much of the
disparity in cardiovascular risk between males and females has been attributed to the role of sexual
hormones, especially oestrogen (4-6). The abrupt decline in cardiovascular health in pre-menopausal
women who undergo surgical menopause further emphasises the association of lack of oestrogen
with endothelial function (7,8). Conversely, the implementation of short-term oestrogen
replacement therapy (ORT) can improve endothelium-dependent vasodilation in post-menopausal
women, and reduce cardiovascular mortality in women with surgical menopause (7-10).
Remote ischaemic preconditioning (rIPC) is one of the most powerful endogenous mechanisms
induced by cycles of brief ischaemia and reperfusion, resulting in protection from subsequent
potentially lethal ischaemia. Thus it holds great potential as an intervention during myocardial
infarction (MI) management using primary percutaneous coronary intervention (11,12). The
technique would allow for major reductions in the reperfusion injury component of infarct size (13).
As generation of the humoral cardioprotective factor in remote ischaemic preconditioning is thought
to be influenced by endothelial function, and rIPC is impaired in those with vascular diseases (14),
we postulated an attenuation of rIPC in post-menopause. Secondly, we proposed a restoration of
rIPC in post-menopause after 3 months of ORT. In order to test these theories, I first required a cell
model of ischaemia/reperfusion injury (I/R injury) upon which to compare levels of cardioprotection.
Our lab had previously used the established cell pelleting technique to simulate I/R injury in rat adult
ventricular myocytes (AVM) (15,16). However, this was a low-throughput method and I required a
higher-throughput model to test large numbers of serum samples collected from human volunteers
who were subjected to rIPC of their upper arm via blood pressure cuff inflation/deflation.
My novel I/R injury protocol for isolated AVM incorporated the use of a hypoxic chamber, removal of
metabolic substrates, and 2-deoxyglucose metabolic inhibitor to simulate ischaemia. Upon
reoxygenation, pyruvate was added to overcome metabolic inhibition. This method allowed for
screening of 24-26 samples per AVM isolation vs 8-10 in cell pelleting, and reduced the amount of
human serum required from 600µl to 250µl. Human serum collected after rIPC, containing the
humoral cardioprotective factor, was incubated with AVM prior to I/R injury. Cardioprotection was
assessed by counting viable and necrotic cells via calcein and propidium iodide fluorescent stains.
Unfortunately, the original proposal to use H9c2 rat cardiomyoblast cell line was not successful due
to the adherent nature of H9c2 during the duration of the hypoxic treatment, and their propensity
to lyse in the flow cytometer. An alternative method of hypoxia/reoxygenation was developed
whereby H9c2 cultured in 96-well plates were serum-starved before conditioning with human
serum. Ischaemia was simulated by hypoxia and use of glucose-free media; reoxygenation took place
with fresh culture media (+10% foetal bovine serum). Assessment of cell viability was then
performed with the MTT assay. Although the H9c2 model provides the best throughput and only
requires 100µl serum it is beset by limitations due to its cardiomyoblast nature.
These 2 hypoxia/reoxygenation methods were compared with the cell pelleting technique as well as
their ability to demonstrate cardioprotection by known cardioprotective drugs – diazoxide and
cyclosporin A. Screening of serum samples from pre-menopausal females (20-30yrs), younger males
(20-40yrs), older males (55-70yrs), and post-menopausal females before and after ORT (40-60yrs)
was completed using the AVM as well as the H9c2 models. Results were compared with those
acquired from the cell pelleting model.
In summary, my data showed a significant loss of rIPC-cardioprotection in post-menopausal females
that was present in pre-menopausal females. This was independent of the effect of aging, as there
was no associated loss of rIPC-cardioprotection in older males vs younger males. There was also no
improvement in cardioprotection afforded by 3 months of ORT in these post-menopausal women.
With regards to the multifaceted pathways of rIPC, it may be that ORT is insufficient on its own to
regenerate the cardioprotective signalling required for rIPC.
The effect of the “timing” hypothesis of hormone replacement therapy was investigated (2,17), and
did not reveal any significant effect of ORT treatment within 5 years of menopause or after 5 years
of menopause. However, my study group was of limited size (12 post-menopause individuals).
Interestingly, serum collected from pre-menopausal women at basal conditions (before rIPC)
demonstrated significant cardioprotection that was comparable to the magnitude of
cardioprotection after rIPC of the upper arm. This was not evident in the younger, age-matched
males.
Unfortunately the basal cardioprotection present in pre-menopausal women was absent in postmenopausal females. This may be the basis for the greater cardiovascular risk and poorer prognosis
of post-menopausal females suffering from acute coronary syndromes.
Although this was a small observational study, it has produced some very insightful results. My only
regret is that it does not delve into molecular pathways or help to elucidate the vital components of
rIPC signalling. However, it has prompted future questions and implications on clinical intervention
for women, especially given that women admitted to hospital with an MI tend to be treated less
aggressively, suffer from poorer prognoses, and are managed according to guidelines which are
based on studies conducted predominantly on male physiology.
This year has been an exciting year and I’m glad to say I haven’t been scared off by research. There
were tough times, but the satisfaction of getting results, whether positive, or negative as in this case,
is intriguing and stimulates further curiosity. I wish to thank you once again for this unimaginably
worthwhile experience.
References
(1) Taddei S, Virdis A, Ghiadoni L, Mattei P, Sudano I, Bernini G, et al. Menopause is associated with
endothelial dysfunction in women. Hypertension 1996 Oct;28(4):576-582.
(2) Vitale C, Mercuro G, Cerquetani E, Marazzi G, Patrizi R, Pelliccia F, et al. Time since menopause
influences the acute and chronic effect of estrogens on endothelial function. Arterioscler Thromb
Vasc Biol 2008 Feb;28(2):348-352.
(3) Celermajer DS, Sorensen KE, Spiegelhalter DJ, Georgakopoulos D, Robinson J, Deanfield JE. Aging
is associated with endothelial dysfunction in healthy men years before the age-related decline in
women. J Am Coll Cardiol 1994 Aug;24(2):471-476.
(4) Sarrel PM. How Progestins Compromise the Cardioprotective Effects of Estrogens. Menopause
1995;2(4):187.
(5) Hashimoto M, Akishita M, Eto M, Ishikawa M, Kozaki K, Toba K, et al. Modulation of endotheliumdependent flow-mediated dilatation of the brachial artery by sex and menstrual cycle. Circulation
1995 Dec 15;92(12):3431-3435.
(6) Townsend N, Wickramasinghe K, Bhatnagar P, Smolina K, Nichols M, Leal J, et al. Coronary heart
disease statistics 2012 edition. London: British Heart Foundation; 2012.
(7) Pinto S, Virdis A, Ghiadoni L, Bernini G, Lombardo M, Petraglia F, et al. Endogenous estrogen and
acetylcholine-induced vasodilation in normotensive women. Hypertension 1997 Jan;29(1 Pt 2):268273.
(8) Rivera CM, Grossardt BR, Rhodes DJ, Brown RD,Jr, Roger VL, Melton LJ,3rd, et al. Increased
cardiovascular mortality after early bilateral oophorectomy. Menopause 2009 Jan-Feb;16(1):15-23.
(9) Lieberman EH, Gerhard MD, Uehata A, Walsh BW, Selwyn AP, Ganz P, et al. Estrogen Improves
Endothelium-Dependent, Flow-Mediated Vasodilation in Postmenopausal Women. Annals of
Internal Medicine 1994 December 15;121(12):936-941.
(10) Bush DE, Jones CE, Bass KM, Walters GK, Bruza JM, Ouyang P. Estrogen replacement reverses
endothelial dysfunction in postmenopausal women. Am J Med 1998 Jun;104(6):552-558.
(11) Sloth AD, Schmidt MR, Munk K, Kharbanda RK, Redington AN, Schmidt M, et al. Improved longterm clinical outcomes in patients with ST-elevation myocardial infarction undergoing remote
ischaemic conditioning as an adjunct to primary percutaneous coronary intervention. Eur Heart J
2014 Jan;35(3):168-175.
(12) Botker HE, Kharbanda R, Schmidt MR, Bottcher M, Kaltoft AK, Terkelsen CJ, et al. Remote
ischaemic conditioning before hospital admission, as a complement to angioplasty, and effect on
myocardial salvage in patients with acute myocardial infarction: a randomised trial. Lancet 2010 Feb
27;375(9716):727-734.
(13) Frohlich GM, Meier P, White SK, Yellon DM, Hausenloy DJ. Myocardial reperfusion injury:
looking beyond primary PCI. Eur Heart J 2013 Jun;34(23):1714-1722.
(14) Balakumar P, Singh H, Singh M, Anand-Srivastava MB. The impairment of preconditioningmediated cardioprotection in pathological conditions. Pharmacological Research 2009 7;60(1):18-23.
(15) Edroos SA, Vanezis AP, Davies MJ, Samani NJ, Rodrigo GC. 108 Remote ischaemic conditioning is
impaired in diabetes. Heart 2012 May 01;98(Suppl 1):A61-A61.
(16) Rodrigo GC, Samani NJ. Ischemic preconditioning of the whole heart confers protection on
subsequently isolated ventricular myocytes. American Journal of Physiology - Heart & Circulatory
Physiology 2008 Jan;294(1):H524-31.
(17) Herrington DM, Espeland MA, Crouse JR, Robertson J, Riley WA, McBurnie MA, et al. Estrogen
Replacement and Brachial Artery Flow-Mediated Vasodilation in Older Women. Arteriosclerosis,
Thrombosis, and Vascular Biology 2001 December 01;21(12):1955-1961.