Glycans and Cardiovascular Disease: Understanding Modifiable Risk Factors and the Power of Lifestyle Choices with Prof. Samia Mora

Podcast published on 5/16/2023 • Show notes written by Vanja Maganjic & Rina Bogdanovic

52 minutes reading time
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Episode summary

It is a widely known fact that cardiovascular diseases are the leading cause of mortality and morbidity in both men and women, which puts an urgency on gaining a better understanding of them. One interesting lens to look at CVDs is through glycans. However, in order to do so, we need to uncover the answer to which came first, the chicken or the egg? Do glycans change in response to the disease? Or are they actively involved in disease development?  To figure this out, we welcomed Prof. Samia Mora to the podcast. Samia is a Professor of Medicine at Harvard Medical School and Director of the Centre for Lipid Metabolomics at the Brigham and Women’s Hospital in Boston, Massachusetts. Her research focuses on risk factors and prevention of cardiovascular diseases (CVDs). Listen in as she introduces us to a number of modifiable risk factors and the cumulative effect both positive and negative behaviours have on future disease risk. 

 

Conversation timestamps

We Discuss:

• Introduction to Cardiovascular Diseases [01:15]
• The Key Role of Inflammation in Cardiovascular Disease Development [02:41]
• The 'Good vs Bad Cholesterol' Perspective: Is It Outdated? [04:05]
• Glycans: Their Role in Predicting Cardiovascular Disease Risk [08:09]
• A Look Back: What We Can Learn from the GlycA Biomarker from the 1980s [10:41]
• Unravelling the Impact of Obesity and Weight Loss on Cardiovascular Disease [15:03]
• The Glycan Dilemma: Do They Change in Response to Disease or Are They Partake in Disease Development? [21:47]
• Risk Factors for Cardiovascular Disease: Modifiable vs Non-Modifiable [25:55]
• Lifestyle Changes: A Proactive Approach to Prevention [31:38]
• The Effect of Weight Loss on Inflammation Levels [40:14]
• How Often Should Blood Lipid Levels Be Measured? [43:17]
• Glycans and Sex-Specific Cardiovascular Risk [48:18]
• Pregnancy and Menopause as Risk Factors for Cardiovascular Disease [50:02]
• Glycans: Effective Biomarkers of Cardiovascular Disease? [58:44]

 

About the guest

Conversation highlights

"So heart attack is the number one killer, unfortunately, in both the US, the UK and globally, across the world. And in fact, it's been increasing over time, because people are now living longer, so they're getting older. And as we get older, the risk of heart attack and stroke increases a lot. It's also the number one cause of death for both men and women. So not just men die from heart attacks and strokes, but also women. And it's across ethnicities, you know, whether you're African American, Black, Asian, Hispanic, you know, different ethnicities, and races all suffer from it as the number one cause of death. So it's a very important disease to study, it's very common, and often there's a long asymptomatic period. So we don't identify people until sometimes it's unfortunately too late. So that's why a lot of my research - I'm interested in trying to identify people having risk factors or increased risk for having a heart attack or stroke at a younger age."

"The most common diseases currently affecting humanity are really cardiovascular disease, cancer, diabetes, and a lot of them can be prevented with a healthy lifestyle. Specifically, heart attack, stroke and diabetes, we can prevent 80 to 90% of these clinical events, purely with lifestyle, but that would be a lifestyle that includes eating a healthy diet such as a Mediterranean diet. The other one is avoiding smoking or not smoking in the first place, avoiding secondhand cigarette smoke, and being physically active."

"However, we also know that we should not treat women or men with oestrogen or progesterone in order to decrease the risk of cardiovascular disease. Because people used to think for the longest time that since premenopausal women have lower risks, especially for heart attacks than men, they must be protected by oestrogen. And while that may be at least partially true, we do know now from several randomised clinical trials that have tested that hypothesis and tried to give postmenopausal women who had low levels of oestrogen, oestrogen and progesterone to get them looking more like pre-menopausal women. We know that that actually increased the risk of cardiovascular events, in particular stroke."

 

Episode transcript

Rina's Intro 

Rina Bogdanovic [00:05] Hello, hello, and welcome back to GlycanHub - the podcast in which we explore health, disease, and longevity through the lens of glycobiology. My name is Rina, and I am your host. Cardiovascular diseases, by which I mean diseases of the heart or blood vessels, are the leading cause of mortality and morbidity in both men and women. Which I realise is a pretty grim statement to start an episode with. But in today’s conversation, we will focus on exploring modifiable risk factors and the cumulative effect both positive and negative behaviours have on future disease risk. We will find out the two leading causes of premature heart attacks in young people, and as you might suspect, both are related to lifestyle. My guest today will introduce inflammation as a key player in the development of cardiovascular diseases and describe the risk in the context of specific health conditions such as obesity, chronic inflammatory diseases, and menopause. We will also discuss the importance of catching cardiovascular disease early and the potential of glycan-based biomarkers. Listen in to discover why glycan conjugates are considered dynamic biomarkers and how they compare to existing diagnostic and prognostic methods. My guest today is a Professor of Medicine at Harvard Medical School and Director of the Centre for Lipid Metabolomics at the Brigham and Women’s Hospital in Boston, Massachusetts. Her research focuses on risk factors and prevention of cardiovascular diseases. A warm welcome to Samia Mora. 

Samia Mora [01:11] Thank you. Nice to join you, Rina. Thank you for the invitation. 

Introduction to Cardiovascular Diseases

Rina Bogdanovic [01:15] Very happy to have you here. I think the most obvious place to start is by asking you what are cardiovascular diseases? And why did you decide to specialise in cardiovascular medicine?

Samia Mora [01:26] So cardiovascular diseases generally encompass heart attack, which is technically referred to as myocardial infarction, and also stroke and dying from those two diseases. So it's the number one killer, unfortunately, in both the US, the UK and globally, across the world. And in fact, it's been increasing over time, because people are now living longer, so they're getting older. And as we get older, the risk of heart attack and stroke increases a lot. It's also the number one cause of death for both men and women. So not just men die from heart attacks and strokes, but also women. And it's across ethnicities, you know, whether you're African American, Black, Asian, Hispanic, you know, different ethnicities, and races all suffer from it as the number one cause of death. So it's a very important disease to study, it's very common, and often there's a long asymptomatic period. So we don't identify people until sometimes it's unfortunately too late. So that's why a lot of my research - I'm interested in trying to identify people having risk factors or increased risk for having a heart attack or stroke at a younger age.

The Key Role of Inflammation in Cardiovascular Disease Development 

Rina Bogdanovic [02:41] Now, when talking about the development of cardiovascular disease, or CVD, for short, I think maybe it's easier to sometimes say CVD in the conversation. What role does inflammation play in this development?

Samia Mora [02:51] Over the past, almost two decades now, we've identified that inflammation is crucial for all aspects of atherosclerosis, which is the underlying biological mechanism behind a lot of cardiovascular diseases, and clinical cardiovascular events - heart attacks and strokes. So we now know, for example, for heart attack, and we used to think it's only just the cholesterol buildup in the arteries that promotes a heart attack. But now we know while that is important, inflammation is crucial as well, and inflammation causes atherosclerosis development, it causes the plaque to rupture, the blockage to explode. And then it's very intimately involved in the thrombus generation. And then also now we know recently, this has over the past, I would say, decade or so that the resolution of inflammation is also really important. And we now know that that's an active process. So it's not just like you have an explosion of inflammation, let's say when you have a heart attack, and then everything just quiets down on its own. No, we know that the body in different cells and different tissues have active ways to actually reduce that inflammation so that the body recovers back to its more normal state.

The 'Good vs Bad Cholesterol' Perspective: Is It Outdated? 

Rina Bogdanovic [04:05] Could you maybe just for our listeners reiterate what is the difference between good and bad cholesterol?

Samia Mora [04:10] It's an important question that you ask because many people think of cholesterol as being this big group of like, good or bad. Well, okay that's another thing. We've also learned a lot more over the past decade or so, some of it from our research showing that having a different pattern of cholesterol can predict cardiovascular disease, and in general, LDL cholesterol is considered a bad cholesterol, because that's the cholesterol carried in these particles. It’s actually carried in the bloodstream in these particles, almost like boats travelling and you have cargo on it and that cargo is the LDL cholesterol on the LDL cargo. Now you have other particles in the bloodstream that also carry cholesterol and also the other important lipid is triglycerides. And that's carried, for example by very low-density lipoprotein LDL particles, which also carry triglycerides. And also you have HDL particles, which is what you mentioned as potentially being the good cholesterol. Although, like I said recently, we now realise it's not like you're either good or bad. We do know that LDL cholesterol, LDL particles, the whole particle, not just the cholesterol carries the VLDL particles, as well as remnants, these kinds of big particles that after we eat a high-fat meal get released in the bloodstream from the intestines. These all promote atherosclerosis, promote clinical cardiovascular events. On the other hand, what we call HDL cholesterol, which used to be called good cholesterol, but now we know there's also different functions to the HDL particle. So some particles have anti-inflammatory functions, depending on the milieu and the function of that HDL. And some HDL particles can actually even promote atherosclerosis, depending on what particle content they have in terms of proteins, in terms of functional capacity of that particle. So it's not just a passive, you know, cholesterol floating in the bloodstream. These particles have active roles. And it's a complex biology that in fact, we're only starting to understand even though we've been studying it for several decades now.

Rina Bogdanovic [06:31] Now, it sounds like we are realising our previous view has been a bit too simplistic. When it comes to cholesterol. 

Samia Mora [06:38] True, I think the bottom line is that you know, cholesterol, having a high level of the bad particles, what we call the LDL, VLDL, or triglycerides, and total cholesterol, all of these should be considered as indicators of risk. Now, it's not the only marker of risks. There are many other risk factors for cardiovascular disease, in particular for coronary events. So heart attacks in particular are promoted by abnormal lipid patterns. But on the other hand, the HDL, if it's low, it is also an indicator that that person is at increased risk, it doesn't necessarily mean that the HDL is causing the risk to be increased, you know like we just talked about inflammation, we know inflammation is very positive. We know LDL particles, including the cholesterol they carry, are also causative of cardiovascular disease. Triglyceride particles and triglycerides themselves, there's more controversy, and HDL particles, there's even much more controversy. So we don't really necessarily know that they play an active role. But we know that there's a lot of mechanisms as HDL is involved in including inflammation, including removing cholesterol from the periphery from the tissue. But in terms of, you know, we can't just say it's very simple for each of us, or it's actually very complex, unlike for LDL cholesterol, or even triglycerides, where we know those are clearly increased risk.

Glycans: Their Role in Predicting Cardiovascular Disease Risk

Rina Bogdanovic [08:09] Now, you have also done quite a bit of research when it comes to glycobiology. And in one of your studies, I'm going to read now what you said. Glycans are described to capture the summative risk information related to inflammation, immune and signalling pathways. Why is this the case?

Samia Mora [08:27] Glycans are kind of the new kid on the block Rina. So I'm a clinical cardiologist, if you ask many clinical cardiologists out there, they may have only heard of glycans in passing or maybe even not at all, because they're really kind of the new kid on the block. We used to think so what are glycans? So glycans are basically these sugar molecules that are attached to either proteins or to lipids, we were just talking about the lipid particles, the LDL, HDL, VLDL particles that carry cholesterol. Well, they also carry on them glycans. Glycans can modify the function of these particles like we were just saying, and we just only started to realise what are these functions. So the glycans were considered before kind of like a sweet dressing, they are sugars. And then the function was not really known for them. But we know also that they have complex structures. So they can be, you can think of it kind of like the Lego puzzles we used to do when we were kids, where you can just kind of add different structures, and if you add the structure, it has a different function. And so now only and really also more recently, past decade or so and even last few years, and this is an area of really active research that we're finding that these glycan structures actually are really important for function. And the function is mechanistic for both inflammation, immune function, cellular biology, cellular mechanisms, molecular pathways, it's really quite extensive, and not just for cardiovascular disease, but also for cancer, for infections, for pulmonary disease, for a variety of systemic diseases and systemic processes. So it's really very critical that we understand their structures and functions because then we can potentially modify them or we can also use them as markers of risk. Like I was saying, we're trying to identify who's at increased risk for having a heart attack or stroke. And we know, we now know that some of these, you know, glycan or sugar molecule additions to proteins and to lipids can actually modify their function and risk.

A Look Back: What We Can Learn from the GlycA Biomarker from the 1980s

Rina Bogdanovic [10:41] Now one such measurement is called GlycA, how does it compare to traditional inflammatory biomarkers?

Samia Mora [10:49] Oh, GlycA was a term ascribed to the signal on the nuclear magnetic resonance spectroscopy signals. So that's often people have heard of NMR, nuclear magnetic resonance, it's a one-dimensional proton of the NMR signal that's used. They noted it back in the 1980s. Bell and colleagues, they're physicists, they noticed this signal in the spectrum of the blood plasma of humans, and they called it GlycA, it seemed at that time, even back in the 1980s. By Bell and colleagues, they found that this particular part of the spectrum was different among people who had inflammatory conditions. So that was kind of the first really report of this GlycA signal. And then, you know, fast forward now to about, you know, two decades later, when this particular, the same signal of the NMR was. Now we were able to measure it basically, on a lot of individuals in a high throughput manner with NMR spectroscopy. This was developed by one of the laboratories in the US that identified this and quantified this GlycA signal, but it's basically based on the same signal that Bell and colleagues used. And once we were able to quantify it using this high throughput technology we can now measure on many hundreds of 1000s of individuals, we had an explosion of studies over the past decades. The first report on the relationship of GlycA with cardiovascular events was actually from our laboratory group, a postdoctoral fellow working with me Dr Akintunde Akinkuolie, who's now a cardiology fellow at UCLA, he was interested in working with me on this project, and we identified that this GlycA signal, measured among 28,000 women and the Women's Health Study that we had followed for over 20 years or so. So we identified that this GlycA signal predicted the risk of future heart attack and future stroke, and these women even followed up to 20 years later. And that was really the first report of its relationship to cardiovascular events. And since then, other studies have confirmed that finding. What's also important Rina, is that while it correlates with other inflammatory biomarkers, you know, when we were talking about how GlycA is a signal related to inflammation? Well, it turns out GlycA is actually not just all the glycan signals, it's reflecting a very specific type of glycosylation pattern of several acute phase reactants. So acute phase reactants are these proteins that increase if there's inflammation, whether it's acute inflammation or chronic inflammation, they can be elevated. And this GlycA signal captures this glycosylation to ascertain the glycosylation pattern from several of these acute phase reactants. For example, clinicians may have heard of Transferrin or Alpha-1 antitrypsin or Alpha-1-antichymotrypsin or Haptoglobin. These are some of the proteins that get captured, the glycosylation of these proteins get captured by this GlycA. And, that seems to be a really good predictor of cardiovascular events. And like I said, while it correlates with inflammation, it moderately correlates with, for example, with high-sensitivity C-reactive protein (hs-CRP) correlation coefficient about 0.3 or 0.4, but it tells a different story. And we found it relates not just to cardiovascular events or heart attack and stroke, but also to diabetes, and importantly to risk of colorectal cancer. That's another important finding that has now also been replicated in other studies, as well as other laboratories have examined how does it change with certain inflammatory conditions like psoriasis or lupus and has also been examined in those conditions as well.

Unravelling the Impact of Obesity and Weight Loss on Cardiovascular Disease 

Rina Bogdanovic [15:03] Talking about chronic inflammatory conditions. Why are people with these diseases at a higher risk of developing cardiovascular disease? 

Samia Mora [15:11] To answer that question, we have to go back to how we were saying how atherosclerosis and inflammation are intimately involved. And as it turns out, you can have basically a normal cholesterol profile, but if you have a lot of inflammation, you're also at quite an increased risk of cardiovascular disease. Now, if you have both high cholesterol, high inflammation and for example, other risk factors that may contribute to both, then you're at a very high risk of cardiovascular disease. So you know, that inflammatory profile is very important, but also the other risk factor profile of that individual. So for example, people who may be smokers, they will have higher levels of inflammation. You know, the more people smoke cigarettes, the more the inflammation. For people who are obese, obesity is now, especially abdominal obesity, visceral obesity is very intertwined with inflammation, in fact, that actually promotes inflammation. And we think now that that's part of the mechanisms of how visceral adiposity and abdominal adiposity relate to increased risk of heart attacks and stroke, especially at young ages. In one study, we found that this increased adiposity is related to greater levels of inflammation and that greater levels of inflammation are related to having more heart attacks and strokes at a younger age. So it's all really intertwined in, you know, sort of complex pathways, but still important pathways that relate to the development of clinically important heart attacks, strokes and other diseases, such as the ones you mentioned, like other inflammatory diseases.

IgG and Cardiovascular disease

Rina Bogdanovic [16:53] Now before we continue talking more about risks, modifiable and non-modifiable risks of cardiovascular disease, I want to first mention immunoglobulin G and glycans. We have mentioned this in quite a few other episodes, but could you tell us what do glycans on an antibody have to do with cardiovascular diseases?

Samia Mora [17:13] I wish I knew the full answer to that, Rina because that's actually an area that we're actively investigating. But I can tell you what we know so far. So we know that IgG for example, glycosylation patterns, again, glycosylation is a certain post-translational modification of the sugar molecules on the IgG. And we know for example, that certain patterns relate to increased risk, and certain patterns indicate more proinflammatory, for example, N-glycosylation of IgG traits correlates with age, they increase as we age, I'm not sure that we know how to necessarily modify them too much, except for that we know that healthier lifestyle, better, more healthy diet, more exercise. Even some studies have shown weight loss can modify these glycosylation or IgG traits. Also, postmenopausal women have a different pattern compared to pre-menopausal women. In terms of the IgG, in particular, IgG glycosylation. I don't think people need to really remember the particular pattern that relates to risk because it's still all being explored. But we do know that certain patches of IgG are key in terms of markers of risk. And, as I said, most likely, they're also mechanistically involved. We can tell that from what we know sort of more basic science experimentation and from genetic studies. We also know we mentioned HDL and LDL, we know that certain patterns of IgG also traits, glycan traits also correlate to the lipids. Some of them also change in terms of lifestyle patterns, so more healthier lifestyle - better traits. Other cardiovascular risk factors also relate to them. For example, blood pressure, we know high blood pressure is one of the strongest risk factors for cardiovascular disease, especially for stroke. And we know that certain patterns of IgG glycosylation, for example, decreased galactosylation, decreased sialylation and fucosylation are actually less sorry, increased sialylation and fucosylation are associated with blood pressure. So, these patterns, do mark certain risk factors, certain diseases, and they're also related to cardiovascular events. In fact, we have a study that we presented with another postdoctoral fellow of mine, Dr Rosangela Hoshi, working together with Dr Olga Demler, myself and Dr. Gordan Lauc group and we presented data from two different studies from the Jupiter population and from the TNT population. So two different studies. One is primary prevention. So these are people who currently have not had any cardiovascular events but have higher levels of general inflammation, high CRP. The other group was TNT, which was a study of people who already had coronary disease, and were being followed for subsequent events. And in these two studies, what we identified was there are certain patterns of the IgG glycosylation traits that really were key in terms of predicting risk. We, in fact, developed a score based on four of them. And the score was associated with about two-fold increased risk of cardiovascular events. And that was independent of the other risk factors, even independent of C-Reactive Protein. So while I mentioned to you that they are related to inflammation, they also seem to identify this, you know, new frontier of pathways that aren't captured by the current clinical markers, because they were also independent of the cholesterol, independent of the other clinical risk factors that we had. We were very excited about these results. And these, in particular, are related to the N-glycosylation patterns of IgG. The study from Jupiter was replicated exactly in TNT. And we're working on the paper that will be submitted to a journal very soon. So we're very excited about these results.

Rina Bogdanovic [21:39] By the time this episode is published, the study might as well, might very well have been published as well.

Samia Mora [21:45] I hope so. 

The Glycan Dilemma: Do They Change in Response to Disease or Are They Partake in Disease Development?

Rina Bogdanovic [21:47] Yes, we'll make sure to link in the description for anyone who is interested. A common question we address in this podcast is what came first, chicken or the egg? Do glycans change in response to the disease? Or are they actually involved in the development of the disease itself? So do glycan changes on IgG which are suggestive of cardiovascular disease, do they occur before the symptoms of the disease begin manifesting?

Samia Mora [22:08] That is, like you said, Rina, a really important question, was it chicken or egg? And sometimes it's actually both. It's a bit of both because often were found in, you know, that it could be like starts early, but then as the disease progresses, it also results in changes. So there are two ways to answer that question. One is to examine sort of relatively healthy people how we were just talking about, how people could have the subclinical cardiovascular disease without having manifested yet clinically, because there's a long asymptomatic phase, of especially coronary disease, but also, you know, stroke. Because atherosclerosis takes time to develop. So there are some ways to look at this. So for example, if you do a randomised trial, and you assign half the population to a certain intervention that modifies the glycosylation pattern, and another t is placebo or control, then you can tell if the intervention in particular, and the resulting changes had an effect. Now, that's a bit harder currently, because a lot of the interventions we have, even those that affect glycans, and glycan structure and function, have also other effects as they may also reduce inflammation, they may modify lipids, they may modify the proteins themselves as well in other ways. But we do know that certain interventions such as weight loss, eating a healthier diet and exercising more do seem to favourably affect glycan traits. We also look to see if statins, you know statins are commonly prescribed medications. And we look to see if statins which are used for cardiovascular prevention, because they reduce clinical events in general by about 20 or 30%, we look to see what's their effect on glycosylation. We look specifically at GlycA levels that we were just mentioning earlier with the NMR signal, but we also looked at HPLC and measured glycans from IgG in the Jupiter and TNT study. And while there were some small changes of GlycA in terms of the statin, it wasn't something to really write home about. It was in the effect of like about 3% change in GlycA that was reduced with the statin and that was a randomised trial. So we could tell for sure it was the effect of the intervention. For the other IgG glycans, there really wasn't much of an effect either or so I think we can say that starters don't have a dramatic effect on glycans. Now, how do other therapies affect - we know also, for example, treatments used as anti-inflammatory treatments, for example, TNF, you know, the TNF modifies TNF-alpha medications inhibitors that are used for treating individuals say with rheumatoid arthritis or psoriasis, those modifying reduced GlycA, and some studies have shown that you can track the GlycA level as a marker of the disease severity. I think this is a really evolving area, and we will know a lot more in the future about, you know, how various interventions affect us. We do know, however, that in many of these studies, the baseline levels of these glycan traits measured at baseline years before the event occurred were predictive. So I think that suggests that they are precursors and mechanistically involved. There's been even genetic support for that. But there's a lot to learn still, and certainly, you know, this is an area of open investigation.

Risk Factors for Cardiovascular Disease: Modifiable vs Non-Modifiable 

Rina Bogdanovic [25:55] Absolutely. You know, I want us to talk more about glycans as biomarkers. But before we do that, let's go back to what you talked about earlier, which was the impact of behaviour on the likelihood of developing CVD? Could you define the difference between modifiable and non-modifiable risk factors?

Samia Mora [26:14] Non-modifiable are things at least currently, like age, we cannot modify age, you know, we all get older, if you're lucky. Other non-modifiable, are, you know, our sex, so whether you are born a man or woman, now while these can be modified in some individuals, but in terms of the risk, or I'm talking in terms of the risk for cardiovascular disease, and also family history, so genetics, which again, maybe starting to be a modifiable trait, because there are now genetic interventions where people can actually intervene and target a very specific change in the genetic information. And, you know, so again, I think the definition of modifiable and non-modifiable may or may not change in the future, but currently, it's age, sex, family history, and then genetics. Now, in terms of modifiable, here's a key point, which I think very few people realise, is that for the most common diseases currently affecting humanity, really cardiovascular disease, cancer, diabetes, a lot of it, the majority of it can be prevented with a healthy lifestyle. Specifically, heart attack, stroke and diabetes, we can prevent 80 to 90% of these clinical events, purely with lifestyle, but that would be a lifestyle that includes eating a healthy diet such as a Mediterranean diet, and lifelong not just eating it for a week or two. And that's going to solve anything. The other one is avoiding smoking or not smoking in the first place, avoiding secondhand cigarette smoke and that would also include e-cigarettes although we know less about that. Having you know, paying attention to weight and preventing weight gain. And also being physically active, of course, there are guidelines for physical activity, but the more we do for physical activity, the more the benefit is, and other things such as how we recognise, like sleep has an important effect that's also part of the lifestyle. So with these lifestyle behaviours, we can really reduce the vast majority of most of these chronic diseases. Now, there would still remain about 10 or 20%, that cannot be avoided, but the vast majority can be prevented. Unfortunately, the hard part of this you may ask - Well, why doesn't everybody just follow then a healthy lifestyle, is because it's really hard to do, especially to do it lifelong. And so that's where a lot of the medication has come in, in terms of being additive to lifestyle, but we should always keep in mind lifestyle. Because with lifestyle, even small changes in lifestyle, we found have really dramatic effects. For example, we conducted a study a few years ago to look to see how does being physically active, how does physical activity help to prevent cardiovascular events? Because we know being physically active prevents about 20 to 30, or even sometimes 40% of cardiovascular events. So we asked how, how does that happen. And we found out that it was actually a combination of different pathways. So some are related to inflammation, such as GlycA or glycans and CRP. But these are just really in this case biomarkers of inflammation. Also it's related to better blood pressure profile, less weight gain, better lipid profile, and in fact, actually, the weight itself had only a small component. So people think - Oh, maybe I'll only benefit from physical activity if I lose weight. It turns out no, you will benefit if you do it consistently and regularly, and it doesn't have to be huge amounts. So you don't have to run a marathon, but you have to be consistent. And if you can do it 80% of the time or so you know, the 80-20% rule, if you can do 80% of the time and be regular, even half an hour a day, five days a week has substantial benefits. If you increase that to about double the amount, so about an hour a day, five times a week, that actually has a lot more benefit. But basically, the idea is that if you're physically active, you can prevent heart attacks and strokes, to some extent. It's not going to cure it, it'll prevent it. And then if you add on top of it, eating a healthy diet, such as the Mediterranean diet, which is a very rich diet full of fruits and vegetables, and olive oil, and nuts, so it's a very varied diet. If you add such a diet, on top of being physically active, on top of avoiding smoking, on top of, you know, general attention to health and avoidance of stress and in sleep, getting enough sleep, etc, then you can really have a substantial effect on not just preventing heart attacks and stroke, but longevity, living longer, living to an older age in a healthier way.

Lifestyle Changes: A Proactive Approach to Prevention

Rina Bogdanovic [31:38] Now thinking of people who might have had unhealthy habits, and want to now improve their lifestyle, or those who might want to continue their unhealthy habits for a while, and then eventually they're going to, you know, change their habits. How does the duration of exposure to modifiable risk factors such as smoking, such as having a higher body weight, relate to the risk of cardiovascular disease?

Samia Mora [32:01] I encourage everyone to do a small change in their lifestyle. For example, for smoking, we know that people who used to smoke cigarettes after about 10 years or so, if they've stopped smoking for about two decades, then their risk is really quite low. And there's a gradual decline in that risk. So for example, if you're still smoking, your risk is high, especially for young people, many people don't know that. But young people have what we call premature heart attacks because heart attacks are supposed to happen in older individuals. So they're supposed to happen in men after age 55, or 60. And then women after the age of 65, or 70. But when we do see them in young people, they're often for two reasons. One is they're actively smoking cigarettes, and the other one is obesity, and diabetes. So a lot of obesity, insulin resistance, and diabetes also increase the risk for premature cardiovascular events. So what I say, some of these lifestyle changes are very challenging, but they add up, so if people can modify, and work on certain small goals, I think the key is not to put a big goal in front of you, but a small goal. So individuals who are smoking for example, if they cut down the number of cigarettes, that will decrease their risk of not just actual heart disease, especially coronary events, heart attacks, but also cancer and other inflammation, other systemic diseases. So it's kind of a cumulative effect. In fact, actually, for a lot of these risk factors, we now know there is this cumulative effect of risk factors, not just for smoking, but for cholesterol, for being physically active or inactive, and it's never too late to change. It's never too late to change. In fact, there are studies showing that if people at middle age, who had a bad risk factor profile in terms of these lifestyles, things we're talking about, if they changed some of those, started instituting healthier behaviours, they actually had improvement in their risk. You know, starting from the time that they changed their behaviour, they're not going to wipe away that effect of 20 or 30, or 40 or 50 years of bad lifestyle, but they can improve it. And that's the key. Because none of us can really wipe away the effects of increased age, ageing is the biggest risk factor for heart disease and stroke and like you said earlier, that's really not modifiable at this point. Maybe sooner we'll be able to modify age, but you can counteract that by having a healthier lifestyle. And while it's difficult, but it's cumulative effects. So basically the bad things accumulate but also the good things accumulate. And they kind of counteract each other in terms of lifestyle. And medications are used for additional prevention. So, for example, statins are now widely commonly used for cardiovascular risk prevention, they reduce risk by about 20 to 30%. If people are physically active, they can reduce their risk by 20 to 30%. And if they do both, then there's more risk reduction. And add on top of that, if they're also eating a Mediterranean diet, which has been shown now from like several randomised trials, not just observational studies, but randomised trials have shown that you have 20-30, up to 50% reduction in cardiovascular events, with Mediterranean diet, even instituted in middle age, even instituted among people with heart disease, never too late to start. So you can reduce your risk, but it takes an effort and the will and the plan and often support. You have to have support from the healthcare system, from your family, from friends, from healthcare providers, etc.

Rina Bogdanovic [36:09] Now, just to stress this more for some young adults, you said that cardiovascular diseases tend to develop over decades. And that's why we associate them as diseases which affect middle-aged or elderly people. But is it safe then to assume that if it takes two-three decades for symptoms to begin manifesting, that people in their early 20s or even late teens are already experiencing developments of cardiovascular disease?

Samia Mora [36:40] What we know is from autopsy studies, they have done autopsy studies, where individuals who are young died from other things such as car accidents or other kinds of accidents. And we know that even when you're really young, like even around the age of two or three or four years old, you already have what's called fatty streaks in the coronary arteries around the heart. So the coronary arteries start developing the very beginning stages of plaque, which ultimately, as we get older, can become manifested clinically as a heart attack or stroke. And many individuals, about half of the individuals in their 20s 30s and 40s already have stage two early plaque formation not just in their coronary arteries, but also in the aorta. And this just basically progresses with age, unless people are doing actively, like I said, the active things to reduce their risks such as active lifestyle, better behaviour in terms of the lifestyle factors, but also potentially medications that can also modify the disease progression. So young individuals should really be aware now that obesity is a big risk factor, especially when it's associated with low HDL cholesterol, high triglycerides and high sugar or the Haemoglobin A1c, which is a test that represents how high your sugar has been over a period of a few months. Those individuals seem to have what we call now metabolic syndrome or the lipid pattern is called Atherogenic dyslipidemia, which is low HDL cholesterol, high triglycerides, and even though the LDL cholesterol may not be elevated, these people have high atherogenic particle burden in them, they have a lot of these particles that can cause cardiovascular disease, and often they get pre-diabetes, diabetes and a lot of premature heart attacks or heart attacks occurring at a young age. And in fact, we in countries were or societies or you know, populations where these risk factors, all conglomerate so when people say smoke a lot of cigarettes, are physically inactive have a high level of metabolically abnormal profiles, like I was just talking about with the obesity, Atherogenic dyslipidemia, visceral adiposity, these populations we see heart attacks occurring in 20-year-olds 30-year-olds, and they don't even have to have a dramatic abnormality in any of these risk factors but often is the combination so they have a little bit of a high blood pressure a little bit of a high triglycerides, a bit of a low HDL cholesterol, they have family history, and they're smoking and voila. You know, you have sort of the setup for a heart attack or even stroke although at that age we often see as cardiologists we see a lot of these premature heart attacks, and it can affect women. You know, women as well as men can be affected when they're young. So the two things for younger individuals to worry about, are smoking and obesity with its concomitant metabolic abnormalities that we were just talking about.

The Effect of Weight Loss on Inflammation Levels

Rina Bogdanovic [40:14] Now, as you said previously, our lifestyle changes can be reflected in the IgG glycans. So, we have talked about IgG glycans, in the context of chronic inflammation, and we have talked in other episodes about biological age. So now taking obesity as an example, for people who undergo weight loss, what happens to their chronic inflammation and their biological age?

Samia Mora [40:38] You can have weight loss now through different ways, right, I mean, so you can have moderate weight loss through lifestyle change, and if people maintain that they can, over time have substantial weight loss. The other way is now medications, we have several medications that are especially now over the past year, we know that some of these GLP-1 agonists. These medications, actually also reduce glucose and Haemoglobin A1c, are used for the treatment of diabetes and can also result in weight loss. And we also know that bariatric surgery can result in dramatic weight loss. In fact, bariatric surgery can result in not just substantial weight loss, but also dramatic, like overnight, almost improvement in inflammation, in lipids. You know, that we're still examining, it's still an area of investigation. So we're finding out what does it do to glycosylation patterns, but overall inflammation and other markers of inflammation are dramatically reduced. We know from smaller studies that have been conducted, that smaller studies that have examined weight loss; because it's hard to do large studies on weight loss over a long period of time because people will generally go back to gaining weight over time. So we know from smaller studies that weight loss experiments have improved, and the glycosylation traits have favourable effects on the glycans. So could that be one of the mechanisms that weight loss is improving risk for developing type II diabetes and, and reducing also risk for heart disease and stroke and other inflammatory diseases that's most likely it is related. And I think an area of active investigation that will be very interesting to examine is in these bariatric surgeries. experiments, we do know also from experiments on Mediterranean diet, that also has positive influence on GlycA, on the glycosylation patterns. So diets is also really important, not just weight loss.

How Often Should Blood Lipid Levels Be Measured?

Rina Bogdanovic [43:17] Just to give our listeners some practical advice, how often should people measure their blood lipid levels?

Samia Mora [43:24] I think it depends on the person and what's their risk profile. So for people who are healthy and have not yet had a heart attack or stroke, the guidelines say you should start around age 20. Some guidelines say you should first measure them around age 10, in order to identify like we're talking about some genetics, some individuals have come from families that have genetic lipid abnormalities that can result in heart attacks, even at a very young age. One of the other lipid biomarkers that we didn't discuss is Lipoprotein(a), which is another lipid biomarker that is also important to check, as that can be genetic. And that can also relate to increased risk of stroke and heart attacks, and checking it once or twice, you know, is sufficient. So basically, if you start, let's say, the first lipid pattern at age 10, you get them checked, and then another time when you're a young adult or aged 20. And then it depends on the risk factor profile, you know, if they generally have little in terms of risk factors, you know, it can be every five years or even every 10 years if they're really good risk factor profiles. If they've already had heart attacks or strokes, then you want to measure them more frequently. If they have diabetes, if they have inflammatory conditions like lupus or rheumatoid arthritis, you want to measure them earlier. Here also I want to say that women often don't get their lipids checked as often as men. And that's a really important thing to keep in mind that you know, we should check them in women and men. That should be equal opportunity in terms of testing, there should also be equal opportunity in terms of treatment and recommendations. Because women also develop heart attacks and strokes, but they just develop them over a longer period of time, thought to be maybe related to being pre-menopausal having some kind of protection, although it's not complete protection, but it has some benefits in terms of the risk factor profile for women. So in general, women tend to get heart attacks and strokes, about 10 years later than men. But that's not a general rule. Some women will get them when they're 20. Some women who have inflammation or who smoke or have diabetes will get heart attacks or strokes at a much younger age. And diabetes is also very prevalent in women as well. So in terms of how often to check them, I think the key thing is also to act on the lipid test, if the lipid test is abnormal, lifestyle is a really important way the lifestyle things we talked about, in fact, AHA calls them now Life’s Essential 8, which includes sleep, and that's one of the eight before they used to call them the AHA Simple 7 which is basically to avoid smoking, or never start smoking, to have a good cholesterol, good blood pressure, good diet, good physical activity, make sure you don't have diabetes, or have a good glucose level. And also to avoid obesity, or have actively tried to lose weight if you're overweight or obese. Those were the simple seven. And now they added on top of it, sleeping, having a good amount of sleep each day is also an important lifestyle. So, for some individuals whose lifestyle is not sufficient in and of itself, then we add medications such as statins, we have other medications now that we also know reduce cholesterol, several non-statin medications that reduce cholesterol to quite substantially low levels. And this is also another tool that we can use to reduce the risk, especially among the high-risk individuals who we know it's just a matter of time before they get the clinical event. And in some people who have also abnormal lipids, you can also consider doing a coronary artery calcium scan, because that can also tell you a picture of the atherosclerosis, the calcifications tend to develop in the arteries that are prone to as an indicator basically, of this person having increased risk for having future risk of coronary event. And in young people, if there's doubt as to whether or not they're at increased risk, you can check your coronary artery calcium scan once, and see if they have already coronary artery calcification at a young age, they're at really high risk. So that can be also another tool that we sometimes get in people, especially if we're trying to decide should we start them on medication? Or, is that the right time to start that patient on medication?

Glycans and Sex-Specific Cardiovascular Risk

Rina Bogdanovic [48:18] Thank you for mentioning women. Because, as you said, cardiovascular diseases are the main cause of mortality and morbidity in both men and women. But we don't hear it talked about much when it comes to women specifically. Now, circling back to also immunoglobulin G glycans, is there a difference in the IgG glycans associated with cardiovascular disease risk between the sexes?

Samia Mora [48:42] In general, the patterns that we've seen, we've been involved in several studies, the studies I mentioned Jupiter and TNT. And then there was another study with our European investigators from EPIC. In general, they tend to be similar. You know, in the EPIC cohort, there was a difference in terms of one biomarker, that seemed to be for one of the glycans, IgG glycans traits, it seems to be different for men and women. But in general, they tend to, if it's abnormal, if it's related to increased risk of CVD in men, it's also similarly in women. So I would say right now, the majority of the data suggests in terms of the cardiovascular risk pattern, that it's a risk factor, if it's a risk factor for cardiovascular disease, it's the same in both men and women. Having said that, there are some differences that are noted in terms of, how men versus women in terms of the IgG glycan patterns. But in terms of their CVD risk profile, it seems to be generally the same, but I think we need more studies. We need more studies with women in it as well. And we're hoping to conduct some of those studies so that we can answer that question more definitively.

Pregnancy and Menopause as Risk Factors for Cardiovascular Disease

Rina Bogdanovic [50:02] You said that women before menopause seem to have higher protection than men. But that changes during the menopausal transition and post-menopause. Could you tell us about what is our current understanding as to why women during and post-menopause have a higher risk for cardiovascular disease?

Samia Mora [50:20] It's an area still that, you know, we know some things about, but we still don't know exactly why. We do know that it may relate to oestrogen levels, being higher among premenopausal women. We also know that lipid pattern profiles change as women go through menopause. But even that change starts even before menopause, like a few years before menopause and even continues for a few years after menopause. You know, the hard part is, it's also hard to differentiate how much of it is the menopause versus how much of it is the ageing because, you know, we were saying ageing is the biggest risk factor for heart attacks and strokes, and how much of it is that. We do know that Lp(a), Lipoprotein (a) levels that I was telling you about which is a marker of increased and it's actually a causative risk factor for cardiovascular disease from genetic studies. We know that that biomarker also changes in the menopause and postmenopause, and it's associated with increased risk. We also know that the glycosylation factor, especially the N-glycosylation pattern of IgG, also changes both with age and also with postmenopausal versus premenopausal women. So it's, I think we don't completely understand the entire picture, but we think it's related to oestrogens and to hormone changes. However, we also know that we should not treat women or men with oestrogen or progesterone in order to decrease the risk of cardiovascular disease, because people used to think for the longest time that because premenopausal women have lower risks, especially for heart attacks than men, that they must be protected by the oestrogen. And while there that may be at least partially true, we do know now from several randomised clinical trials that have tested that hypothesis and tried to give either postmenopausal women who had low levels of oestrogen, they tried to give them oestrogen and progesterone to get them looking more like pre-menopausal women. We know that that actually increased risk of cardiovascular events, in particular for stroke. Now there has been debate as to whether if you started those hormones earlier, like let's say you started those hormones not like 10 years after menopause. But let's say you started them right, as a woman who's going through menopause, there's some data to suggest maybe they could be beneficial there for cardiovascular disease. But the general recommendation right now is do not use oestrogen or progesterone in order to decrease the risk of a woman postmenopausal woman. We give the same recommendations to postmenopausal women as we do to manage a woman which is to focus on addressing their cardiovascular risk factors leading a healthier lifestyle for the risk factors we talked about. And also to pay attention, some of them may not have ever had a lipid measurement. So they should definitely have a lipid measurement, have their inflammation level checked to have their Lipoprotein (a) checked. Because those are ways that we can identify if they're at risk for cardiovascular disease. There are a couple of other items that are important for women that I want to mention that I haven't mentioned yet. And those are recognised now by the American College of Cardiology and American Heart Association as of couple of years ago. And this is an area again of active investigation that we hope we'll see a lot more in the future. These are specific for women - is to ask about their pregnancy history and about their menopause history. So a key question to postmenopausal women is when did you go through menopause? So we now know that earlier menopause, what we call premature menopause below age 40, most women go through menopause in their 50s, like 53-55 around that age, but if they had it at 40 or less, that alone is an increased risk for cardiovascular disease. We also know that their pregnancy history of those women who were pregnant, we should ask them about their pregnancy history because that can give us a glimpse of their future risk. If you're trying to predict for that woman her future risk and she's now only 30 or 25. But let's say she was pregnant and had preeclampsia, which is where the blood pressure increases, or she actually had eclampsia where you have also not just that very high blood pressure but also affects the kidney function and the platelets, or if they had pregnancy related gestational diabetes, their sugar increased as a result of the stress of pregnancy, that is really a marker for future development of diabetes. And as we said, diabetes is associated with increased risk of coronary events prematurely. And you can ask her also how were her periods? Did she have regular periods, women who have irregular periods, are also at an increased risk of future cardiovascular events, thought to be maybe because of this polycystic ovarian syndrome. This pattern where these women have irregular periods, and also may have hirsutism and abdominal obesity. So these are other areas that we should in particular for women, especially if your OBGYN or primary care providers should ask women and women should be aware of those risk factors because if they do have them, they should pay much more attention and have their risk factors monitored much more closely, when compared to somebody who's generally healthy. And of course, family history is really important. You know, we should ask men and women did they have, do they have a first-degree member in their family, especially like a parent or a sibling, or of course, a child who had premature heart disease or premature stroke, and that's generally considered premature is if it's younger than age 55 for a man or younger than age 65 for a woman.

Rina Bogdanovic [56:43] What I frequently hear is that HRT can be used as a preventative measure for many chronic conditions. So are you saying that it actually might do the opposite when it comes to cardiovascular disease?

Samia Mora [56:53] Absolutely. That's why I mentioned earlier we need to do randomised trials where you actually test an intervention because sometimes it can be confusing from observational studies. But yes, we now have definitive evidence that we should not be giving oestrogen and progesterone to postmenopausal women to reduce their risk of cardiovascular disease. Now, they may be given for other reasons. For example, a recently, postmenopausal woman or a woman going through menopause, who has significant hot flashes or significant symptoms, so she cannot function, they can be given for a short period of time, but they should not be given for prevention of cardiovascular disease. In fact, they actually increased risk, in particular for stroke, but they also increased risk of thromboembolism, chronic venous thromboembolism, so clots in the legs that go to the lung, as well as also that risk is increased. And we know that they do not prevent, they do not let these women live longer. So again, there's no real reason to give them for that purpose. If they're given there, they may be given for other purposes, such as symptoms of a woman who has suffered a lot of symptoms from menopause. And we should always focus on the risk factors that we know are related to cardiovascular disease.

Rina Bogdanovic [58:16] Yeah, thank you. I'm very glad we brought this up. This was definitely an area that's I think, becoming more and more of a hot topic recently. And I think it's very good to have all evidence shared very clearly, I think a very good place to finish this conversation is by coming back to glycans as biomarkers. Now, what do we mean when we say the glycan conjugates are very dynamic biomarkers? What is the advantage of this?

Glycans: Effective Biomarkers of Cardiovascular Disease?

Samia Mora [58:44] What we mean is that they change when they get modified by various processes. They're not static, it's not like you just have this pattern that stays there, and is there forever? They can be very dynamic. It's a dynamic process. That is modifiable by different processes, whether it's inflammation, whether it's immune function, whether it's, you know, age, whether it's a cardiovascular risk factors, whether it's medications, whether it's related to the disease itself, you know, like the Chronic Disease Burden, can also be related to it, whether it's related to gender or sex and men versus women or ethnicity. It's dynamic. It's not a not a single measure. So what we do know, though, is that the tests that we have currently available have been rapidly getting developed more and more advanced technology. That's I think key because we cannot use something as a biomarker unless you can measure it. And it has to be measured reproducibly. So it has to be if I can measure it in this person, I want to know that, if I measure it again with the same law, that would give me the same result, if nothing else has changed, I want to know that if I measured in my country, and somebody measures a halfway across the globe, that we’ll both get the same results. So what we call standardisation. So you want a lab test that's a good biomarker, something that is easy to measure, is not so expensive, it can be widely available, not just using advanced technologies, but also that could be that is standardised and that it has shown to be reliable, in terms of the measurement. Once you have all of those, then you want to know okay, well, is that a good biomarker for risk? If it's a good biomarker for cardiovascular risk, that should also include not just that it's related to cardiovascular disease risk, that, for example, high level is associated with an increased level or vice versa. But you also want to know that it's really giving you information that's independent of an additive to the other clinical risk factors. Because we already measure a lot of things. We measure people's blood pressure, we measure people's cholesterol and their sugar, we measure kidney function, we measure a lot of things that can tell us about somebody's risk. We also can ask them questions about their lifestyle that also tells us about their risks, we ask them questions about their genetics, their family history, so we'd only want to really add another biomarker, to recommend measuring it if it's been consistently shown to be related to cardiovascular risk, whether it's a positive relationship or an inverse relationship, it doesn't really matter as long as it's related. And that has to be reproducible. It has to be seen not just in one population, but in multiple populations. And it has to be a reliable test that we know we can use, we can measure it with accuracy, and it's standardizable so that people across the globe can be measuring the same thing or with different labs. Those are the key things that make a biomarker, a good measure. And some of the glycan traits that we talked about, have very promising potentials as clinical biomarkers, because the technology has really dramatically improved over the past few years, so that now we can measure it at relatively low cost, it can be reproducible. We need to work more on standardisation, that is an important area that we need to work more on, which I think is an area that people are the labs, you know have to contribute to that. And in terms of the studies, we now have really an increasing body of studies, that is showing that these biomarkers, for example, GlycA or the IgG glycosylation trace that we talked about are related to cardiovascular risk, independent of the standard risk factors that we know. So, therefore, it would make sense to measure it. The other reason to measure these biomarkers could be for somebody who, say you're measuring them to see your response to therapy. If the therapy affects these measurements, and it can help you to let's say back off on the therapy or to give more therapy that is another use for biomarkers in cardiovascular disease clinically, and those areas I see as being two that are really important to develop further.

Rina Bogdanovic [01:03:43] Sounds like glycans are up for the challenge of becoming additional cardiovascular disease biomarkers. But I think a good final question would be, what do you think should be the next steps in research and clinical practice regarding glycan biomarkers for cardiovascular disease? 

Samia Mora [01:03:58] For research, what we really need is more studies in different populations by different ethnicities that incorporate a larger number of cardiovascular events, because we have now quite a few smaller studies, but we need, you know, if you look at the genetic studies, for example, they use hundreds of 1000s of cardiovascular cases. So it would be great to get to that level of studies that we can say, we have a lot more information. In terms of clinical, I think we can use it even currently for example, the GlycA test is provided in the US currently as part of a panel that's not very expensive, and it can be used in clinical practice as a biomarker for inflammation. IgG patterns in certain diseases can also, you know, the glycans traits can be used. We didn't discuss some of the renal and other specific diseases that have glycosylation abnormalities. We were focusing more on cardiovascular disease today. But those are areas of active investigations and I think also standardisation of the testing, we need to have more laboratory led standardisation efforts that could be used worldwide so that this can really end up being a test that is used worldwide.

Rina Bogdanovic [01:05:31] Excellent. Thank you very much, Samia, thank you for coming.

Samia Mora [01:05:33] Oh, thank you so much Rina, for the interview and for highlighting these important lifestyle, biomarker mechanistic studies that have given us a lot of information about cardiovascular disease, development and prevention. Thank you.

Rina’s Outro

Rina Bogdanovic [01:05:52] Now speaking to our listeners, I hope this conversation allowed you to better appreciate the cumulative effect of behaviour on future health. It certainly made me question what all of the small lifestyle choices I make on a daily basis will add up to over time. Samia’s research was supported by research grants from the National Institute of Diabetes and Digestive and Kidney Diseases. and the National Heart, Lung, and Blood Institute. She wished to use this opportunity to thank them. If you would like to access more information about this conversation and Samia’s previous work, follow the link in the description to the show notes for this episode. Equally, if you want to find out more about GlycanAge, head on to glycanage.com where you can access a whole list of our scientific publications, blog posts, testimonials and of course this is where you can order your GlycanAge test kit. Watch out for our next episode where I will be joined by Ania Halama and Karsten Suhre, scientists from Weill Cornell Medicine College in Qatar whose research focuses on metabolomics. We will explore the challenges and benefits of mapping out human metabolic individuality and discuss the place of glycans in multi-omic research. Please don't forget to leave ratings and reviews for this episode and engage with us on social media. Thank you for listening and have a great day.

 

Disclaimer

Please be advised that this show is for information only and should not be considered as a replacement or equal to medical advice.