Circulation July 23, 2019 Issue

Dr Carolyn
Lam:               
Welcome to Circulation on the Run, your weekly podcast summary
and backstage pass to the journal and its editors. We're your
co-hosts, I'm Dr Carolyn Lam, Associate Editor from the National
Heart Center and Duke National University of Singapore.


Dr Greg
Hundley:            
And I'm Greg Hundley, Associate Editor at the Pauley Heart Center
at VCU Health in Richmond, Virginia.


                                               
Well Carolyn, did you ever wonder whether cardiovascular drug
effects could be investigated through natural variation in the
genes for the protein targets? In our feature discussion today,
investigators from the British Isles, Germany, and the United
States use this approach to explore the potential side effects
and repurposing potential of antihypertensive drugs. Sound
interesting? Well listeners, we look forward to the results later
in our program, but Carolyn, how about we chat about some of the
other papers in this issue?


Dr Carolyn
Lam:               
You bet Greg. So, have you ever asked yourself "What is the role
of protein glycosylation in regulating LDL metabolism?"


Dr Greg
Hundley:            
That was going through my mind when we were playing basketball
just the other night.


Dr Carolyn
Lam:               
Well this is truly a great study from Dr Holleboom at Academic
Medical Center Amsterdam and Dr Lefeber from Radboud University
Medical Center, both in the Netherlands. And their colleagues
will study 29 patients of the two most prevalent types of Type 1
Congenital Disorder of Glycosylation, and these are the ALG6 and
PMM2 types. They also study 23 first and second-degree relatives
with a heterozygote mutation and measured their plasma
cholesterol levels. LDL metabolism was studied in three cell
models. They found that patients with type 1 congenital disorder
of glycosylation have hypobetalipoproteinemia through increased
LDL receptor expression. Carriers of the mutation in
glycosylation enzymes affected in this syndrome had decreased LDL
cholesterol levels compared to controls, and defects in
glycosylation enzymes could play, therefore, an important role in
LDL cholesterol metabolism.


Dr Greg
Hundley:            
Boy, this is pretty insightful I think, Carolyn. So, what are the
clinical implications?


Dr Carolyn
Lam:               
Well, given that LDL cholesterol was also reduced in a group of
clinically unaffected heterozygotes, the authors propose that
increasing LDL receptor mediated cholesterol clearance, by
targeting N-glycosylation in the LDL pathway, may therefore
represent a novel therapeutic strategy to reduce LDL cholesterol,
and of course prevent cardiovascular disease.


Dr Greg
Hundley:            
Very interesting work. You know, we just keep learning more and
more about LDL. I'm going to switch and jump back with
Empagliflozin. And this is a study in diabetic mice that really
has an interesting in-vivo imaging component. As an imager, I was
really excited about this. The article is from Dr Kengo Kidokoro
from Kawasaki Medical School. And we don't often talk about it,
but listeners, if you have a chance, there's a very interesting
video-enhanced file associated with this article, and if you can
download it, it's really just so cool with multiple image clips
demonstrating an operative mechanism of SGLT2 inhibition on renal
function. And it really gives us an opportunity to revisit renal
function.


                                               
Quick quiz Carolyn. In diabetic kidney disease, is glomerular
hyperfiltration good or bad?


Dr Carolyn
Lam:               
Bad.


Dr Greg
Hundley:            
Yeah, absolutely. So, hyperfiltration is characteristically
observed at earlier stages of diabetic kidney disease and
involves activation of the renin-angiotensin-aldosterone system
at the efferent arteriole and tubuloglomerular feedback
mechanisms, especially at the afferent arteriole. So, as they go
through this, just picture in your mind that glomerulus and
afferent is arriving, and efferent is leaving.


                                               
So, SGLT2 upregulation in diabetes is thought to play an
important role in TGF signaling by increasing sodium reabsorption
at the proximal tubule, thereby decreasing distal delivery to the
sodium sensing macula densa at the juxtaglomerular apparatus.
This decline in distal sodium delivery is interpreted as a
decline in effective circulating volume, leading to inappropriate
afferent vasodilation in an effort to preserve intra-glomerular
pressure and GFR.


                                               
In diabetes, these TGF effects lead to intra-glomerular
hypertension and hyperfiltration. You got that quiz right,
Carolyn. Which promotes diabetic kidney disease progression and
impaired kidney function, ultimately increasing overall
cardiovascular risk and mortality. Conversely, blocking SGTL2
pharmacologically reduces renal hyperperfusion and
hyperfiltration in animals and humans, which may preserve renal
function, thereby reducing risk associated with diabetic kidney
disease progression.


Dr Carolyn
Lam:               
You know what, Greg? I kind of had an unfair advantage in this
quiz. I work with a lot with the SGLT2 inhibitors, but I just
love that you asked us to picture it and look at that video.
Anyways, so this article really allows us to review SGLT2
inhibition at the glomerular level, which is truly hot. So, tell
us what did they find?


Dr Greg
Hundley:            
So, this is the first report of changes in renal hemodynamic
function by SGLT2 inhibition using direct in-vivo visualization
techniques in a diabetic animal model. The videos, they're
spectacular, and they're excellent so that you can download them
for educational purposes. Afferent arteriolar vasoconstriction,
and reduced hyperfiltration occurred within a few hours after a
single dose of a SGLT2 inhibitor. And Adenosine signaling,
through tubuloglomerular feedback, is a key pathway to prevent
diabetic hyperfiltration via SGLT2 inhibition.


                                               
Clinically, Carolyn, now I know you would ask me about that, so I
got ready, this study highlights another potential mechanism for
the benefits of SGTL2 inhibition. The SGLT2 inhibitor-related
mechanism's responsible for reducing cardiovascular risk in
clinical trials may be due to protection against diabetic kidney
disease progression, thereby attenuating risk factors for heart
failure, such as volume overload and hypertension.


Dr Carolyn
Lam:               
Ah. That is just so cool, and really just so consistent with the
clinical data that's emerging too. Thank you, Greg. So, have you
ever asked yourself this other question, what role do platelets
play in ischemia reperfusion injury? So, I'm not going to quiz
you. I'm actually kind and loving and a good person. And so, I
will tell you about ischemia reperfusion injury, which is a
common complication of cardiovascular disease.


                                               
Now, resolution of the detrimental effects of ischemia
reperfusion injury generated prothrombotic and proinflammatory
responses, is essential to restore homeostasis. Now, although
platelets are known to play a crucial role in the integration of
thrombosis and inflammation, their role as participants in the
resolution of thrombo-inflammation is really under-appreciated.
And hence, this other paper that I chose today, and it's from Dr
Gavins from Louisiana State University Health Sciences Center
Shreveport, and her colleagues, who used pharmacological and
genetic approaches, coupled with murine and clinical samples to
uncover key concepts underlying this role for platelets.


Dr Greg
Hundley:            
So Carolyn, what did they find?


Dr Carolyn
Lam:               
Well, they found that exacerbation of thrombo-inflammatory
responses occurred in ischemia reperfusion injury mouse models of
middle cerebral arterial occlusion, as well as lower plasma
levels of the anti-inflammatory pro-resolving protein Annexin A1.
And this was a lower plasma level of this Annexin A1 among
patients with acute ischemic stroke.


                                               
Administration of Annexin A1 promoted cerebral protection against
thrombo-inflammation and the development of subsequent thrombotic
events post-stroke. Annexin A1 was also able to reduce platelet
activation and thrombosis, via the suppression of integrins. So,
overall, these data reveal a novel multi-faceted role for Annexin
A1 to act both as therapeutic and prophylactic drug via its
ability to promote endogenous pro-resolving anti-thrombo,
anti-inflammatory circuits in the cerebral ischemia reperfusion
injury. And collectively, these results further enhance our
understanding in the field of platelet and ischemia reperfusion
injury biology.


Dr Greg
Hundley:            
Oh wow. So, another important insight from this author group on
platelet activation and thrombosis in key clinically relevant
syndromes. Well, my last paper is going to be talking about a
risk prediction score for life-threatening ventricular
tachyarrhythmias. And they're going to study this in
laminopathies, and the lead investigator is Dr Karim Wahabi from
Cochin Hospital in France.


                                               
To estimate the risk of life-threatening ventricular
tachyarrhythmia in patients with LMNA mutations, and thus select
candidates for implantable cardiac defibrillators, the
investigators evaluated 444 patients of about 40 years in age in
a derivation sample. And then, 145 patients that are about the
same age, 38 years, in a validation sample, for the occurrence of
a) sudden cardiac death or b) ICD-treated or hemodynamically
unstable ventricular tachyarrhythmias.


Dr Carolyn
Lam:               
Oh. Very important. These laminopathies are really not that
uncommon. So what did they find, Greg?


Dr Greg
Hundley:            
Carolyn, predictors of events included male sex, non-missense
LMNA mutations, first-degree and higher AV block, non-sustained
ventricular tachycardia, and LVEF. The authors developed a new
score to estimate the 5-year risk of life-threatening ventricular
tachyarrhythmias in patients with LMNA mutations. And compared to
the current standard of care, the proposed risk prediction model
offered more accurate prediction of life-threatening ventricular
tachyarrhythmias, and correctly re-classified almost 30% of the
patients in the study.


                                               
Nicely, the authors have made this available, and the score can
be derived from readily collected clinical and genetic parameters
and estimated using an online calculator that's provided in the
journal. But, it's https://lmna-risk-vta.fr.


                                               
Future prospective studies should focus on the estimation of the
clinical benefit conferred by the use of this score in terms of
sudden cardiac death prevention.


Dr Carolyn
Lam:               
That is super cool, Greg. But, I am so excited now to move to our
feature discussion. Shall we?


Dr Greg
Hundley:            
You bet.


Dr Carolyn
Lam:               
Can we use natural variations in our genes for the protein
targets as a way to look at cardiovascular drug effects? Man,
this is going to be such an important and exciting discussion,
because this is what our feature paper talks about. I am so
pleased to have with us our corresponding author, Dr Dipender
Gill from Imperial College London, as well as our Associate
Editor, Dr Wendy Post from Johns Hopkins.


                                               
So, first of all, Dipender, please, could you give us a
background on what you did? This is really very novel in
approach.


Dr Dipender
Gill:              
It was also a lot of fun to conduct. I think, currently, we're
living in an era where there's been a recent explosion in the
availability of genetic data, and this really inspired us to
think about how we could use that to learn more about commonly
prescribed drugs. The implementation of genetics, or genetic
variance, to study drug effects isn't entirely novel. It's
actually been undertaken for some years now.


                                               
Most of the work has been related to lipid lowering drugs, for
example, statins, where people can take genetic variance, or
versions of genes, corresponding to the drug effect, and study
these to investigate what effects these drugs might have, both on
the intended target, but also potential side effects. To my
knowledge, this hadn't previously been done for anti-hypertensive
drugs. But yet, the data for this was available. And therefore,
we thought that actually we could very well go ahead and do this,
and perhaps find some interesting things.


Dr Carolyn
Lam:               
Oh, that's so interesting thing, Dipender. You know, there was
this term in your abstract, and mentioned multiple times,
Mendelian randomization. Now, for those of us that don't think
about this every day, could you tell us a little bit what that
means?


Dr Dipender
Gill:              
Yeah. So, I'll actually give a little bit of background. One of
the main limitations of traditional epidemiological research is
that any association, it's sometimes difficult to infer
causation. They can be confounded by environmental factors,
lifestyle factors. In the Mendelian randomization technique, what
we do is we use randomly allocated genetic variants to study the
effect to an exposure.


                                               
So, we select these genetic variants because they are related to
the exposure of interest. And because these genes are randomly
allocated at conception, they're not subject to confounding from
environmental or lifestyle factors. Whether you have a gene or
not, is not necessarily related to your lifestyle or your
environment. And therefore, the association of these genetic
variants with certain outcomes isn't subject to confounding.


Dr Carolyn
Lam:               
That makes so much sense, and I suppose that, not to allow cause
and effect to be determined. So please, tell us, in this
particular case of the anti-hypertensive drugs, what did you do
and what did you find?


Dr Dipender
Gill:              
First, we decided specifically which drugs we wanted to look at,
and we thought, actually, let's start off with the most commonly
prescribed anti-hypertensive drugs. So, we short-listed these
based on recent consensus guidelines, and we looked at ACE
inhibitors, beta-blockers, calcium channel blockers, thiazide
type diuretics. And then, we went back to various online
databases to identify which genes correspond to the target
protein of these drugs.


                                               
We took these genes, and we then identified genetic variants at
their specific genetic loci, their specific region of the genome,
and we identified the variants in these regions that were also
related to systolic blood pressure. And in this way, we inferred
that genetic variants, at the protein coding targets of these
genes, that were also related to systolic blood pressure, likely
represented the effect of variations in these proteins that also
implicated blood pressure, and therefore, could serve as proxies,
or instruments, to study the effect of these drug targets.


                                               
We then went ahead to validate the selection of these genetic
variants by forming Mendelian randomization, and specifically, we
checked whether people that have genetic variants that correspond
to, say, ACE inhibitor activity, or beta-blocker activity, or
calcium channel blocker activity, if they also have
correspondingly lower risk of coronary heart disease and stroke,
to the same degree that we would observe in randomized control
trials against placebo.


                                               
And indeed, we found that actually, the results were fairly
similar, and this gave us confidence. And studying these genetic
variants that mimic the effect of these drugs could be used as a
proxy or as a surrogate to study their clinical effect of taking
these drugs. So, that was the first phase.


Dr Wendy
Post:                
Dipender, congratulations to you and our team. This is a really
exciting paper, and the editors were especially interested in the
novelty, and the potentially impactful findings, especially of
the second part of the study, which I think you'll describe
briefly next. And that was using an approach that many who are
listening may not have heard about too much before called PheWAS,
or a phenome wide association study. And maybe you could tell us
briefly what you found in that part of the analysis.


Dr Dipender
Gill:              
The first part, it was very cool, because it allowed us to
identify versions of genes that corresponded to the effect of
these drugs. But in itself, it didn't tell us anything novel. It
didn't tell us anything new. So, the real question was, how could
we use this new information to make progress towards helping
patients? So, we went back and we thought, "So okay." So, we knew
that these drugs are used for certain conditions already to
prevent heart disease, to prevent stroke.


                                               
But, what about their side effects? What about their repurposing
potential? How could we use our new approach to study that a
little bit more carefully? As you alluded to, when we used this
new technique, relatively new technique called phenome wide
association study, and we essentially investigated the
association of our genetic variants for each respective
anti-hypertensive target with all clinically relevant outcomes
throughout the phenome, using the UK bio-back cohort, which was
the main population used for this PheWAS, this phenome wide
association study.


                                               
We were actually able to rapidly investigate over 900 disease
outcomes, and their association with our genetic risk score for
these drugs. And this was very exciting for us, because it
allowed us to very rapidly, efficiently, and cost-effectively
explore the potential repurposing opportunity and side effects of
these very commonly prescribed drugs, which to our mind, offered
significant advantage over previous approaches.


                                               
We all know that sometimes randomized control trials can be very
expensive and time-consuming, and of course, traditional
observational research can be limited by reverse causation,
assessment-vise confounding. And so, what we were able to do here
had several important advantages, and not to mention the
efficiency by which it allowed study of these outcomes.


Dr Wendy
Post:                
Dipender, tell us what you found in your PheWAS study.


Dr Dipender
Gill:              
We identified genetic variants for 3 commonly prescribed
anti-hypertensive targets. The first were ACE inhibitors, second,
beta blockers, and the third were calcium channel blockers. When
performing PheWAS for all of these drug targets, we identified
associations with common cardiovascular disease that are related,
or implicated in hypertension, specifically hypertension itself,
but also circulatory diseases, things like atrial fibrillation,
coronary heart disease. They all came up.


                                               
And this actually gave us a lot of confidence because that's
exactly what we'd expect. We know that these medications prevent
or reduce risk of these diseases, and therefore, this served as
kind of a positive control that our approach was doing it what it
was supposed to do. The novel finding came when we investigated
the genetic risk score, or the genetic variants for calcium
channel blockers, in this PheWAS approach.


                                               
And we actually identified an association which we weren't
expecting. We showed that blood pressure reduction through the
genetic risk score for calcium channel blockers was an
association with an increased risk of diverticulosis, a condition
not conventionally thought to be associated with blood pressure.
We were very excited and interested by this, and we went on to
investigate it further using some other techniques as well.


Dr Wendy
Post:                
The really impactful part of this, many things, but especially
this association with diverticulosis. So, maybe you can briefly
summarize what you think the potential clinical implications are,
and what the next step should be.


Dr Dipender
Gill:              
The first question we had was whether this was related to blood
pressure alone, the effect of calcium channel blockers, or
perhaps some other effect of these drugs. We investigated the
genetic risk score for systolic blood pressure generally and
found that this itself wasn't associated with risk of
diverticulosis, which suggested that the effect isn't really
mediated by blood pressure alone, but it's some other property of
calcium channel blockers.


                                               
We know that sometimes calcium channel blockers can be associated
with constipation, and it may be through this mechanism that
they're having consequent effects on risk of diverticulosis.
Other possible mechanisms might be through effects on blood flow,
through the vasa recta in the bowel. But, what was very
interesting was that we went forward with this finding, and
investigated, observed, drug use in the UK bio-bank.


                                               
Specifically, we looked at people taking non-dihydropyridine, and
dihydropyridine calcium channel blockers at baseline, and found
that those taking non-dihydropyridine calcium blockers only were
known to have a higher risk of diverticulosis as compared to
those taking other anti-hypertensive classes, which further added
support for our findings. The interesting point here is that
looking at the genetics doesn't allow us to discriminate between
these drug classes.


                                               
That was only possible with the observed data, and that was
because the genes for these drug classes were the same.


Dr Carolyn
Lam:               
Well, congratulations. Wow. I'm just so intrigued listening to
all of this. Wendy, I would love if you could help put all of
this in context for us. The US, the novel information, and the
approach that could potentially go way beyond just
anti-hypertensive.


Dr Wendy
Post:                
So, this is a very exciting new approach to doing genetic studies
that can help us to understand potential targets for therapy in
the future, and understanding more about causality, which as
Dipender explained, can sometimes be confusing, as it may be
confounded by environmental factors. So, using these genetic
approaches through Mendelian randomization, and what we heard
about today, which is PheWAS, or phenome wide association study,
we can learn much more about how the potential observational
analyses can be related to new discoveries through mechanisms, or
potential side effects, as we heard about here of calcium channel
blockers.


                                               
So, wanted to congratulate Dipender again with his impactful
paper here.


Dr Carolyn
Lam:               
Thanks, Wendy. And then if I could, I'm just going to steal
minutes here, because this is so interesting. Where do you think
the field's going to go next? And Dipender, with these findings
of diverticulitis and diverticulosis, what next? How do we apply
this?


Dr Dipender
Gill:              
There's 2 main points to cover here. The first is what we do
specifically with the findings we got for calcium channel
blockers and diverticulosis. I should emphasize that on their
own, I don't think that this should currently change practice.
But, I think it should inspire and capitalize further research
into this association. If we're able to replicate and validate it
further, then perhaps there might be some implications for the
drugs that we prescribe with patients at risk of diverticulosis.


                                               
The second point I wanted to make is more generally, what does
this mean for research, and particularly, genetic research. I
think we're living in very exciting times, and there's a lot of
really great work that's going to come out using these types of
approaches. I think 2 areas that we could expand further is what
else we can do with our genetic instruments, or our genetic
variants that proxied these drugs. How do we look at other
targeted refocusing potential? Can we try and explore other side
effects? Can we investigate efficacy for other disease outcomes?
Specifically, for these anti-hypertensives.


                                               
And the other thing is, which other drugs can we identify genetic
variants to proxy? We've been thinking about looking at diabetes
medicines. There's a variety of other drugs that correspond to
specific gene targets, and proteins. And in theory, these could
also be studied using genetics. So, there's a lot more work to
come out from this.


Dr Carolyn
Lam:               
Thanks so much, both of you, for joining us today. This was just
such an exciting discussion.


                                               
Thank you for listening to Circulation on the Run. Don't forget
to tune in again next week. This program is copyright American
Heart Association, 2019.