Designed By Nature

Recently I wrote an article exploring what could be at the root cause of atherosclerosis. After the publication a comment pointed to the macrophage and cholesterol not being discussed. I didn’t do this because the root cause brought me to a point before macrophage and LDL-cholesterol comes into the picture.

But it got me thinking… what about those macrophages? Why do they take up that cholesterol? What are they supposed to do with it? Why aren’t they doing anything with it? Is it normal for them to just sit there and engulf the LDL cholesterol? Do they take up the LDL by accident and this disables them to function?

Nobody seems to talk about the role of macrophages, why is that? Do they do everything as is supposed to in the development of atherosclerosis? How is the LDL causing an issue here? Is it the fault of cholesterol itself that it gets stuck there? Or is there more to it?

They do correlate well with the lipids and severity of the atherosclerotic plaque. Could there be a problem with the macrophages?

“Relation of Plaque Lipid Composition and Morphology to the Stability of Human Aortic Plaques” https://www.ahajournals.org/doi/10.1161/01.ATV.17.7.1337

I have read my fair share of research in the last 3 years and this gets remarkably little attention but there is no lack of research. As you can see there are a lot of questions to raise and clear up so it is time for another dive into an area where I know nothing about, exciting!

My very basic understanding about macrophages started with them being cells that can kill pathogens and malfunctioning cells by taking them up and dismantling them, releasing the pieces as debris for recycling or elimination by the liver.

Result

I want to start with the result first because there is a lot of ground to cover. The full article is quite detailed so for those who cannot spare the time, here is the result in brief.

Hypoxia causes macrophages to become immobilized. As a first step they become a pro-inflammatory glycolytic M1 type in order to stimulate growth. These M1’s prefer the uptake of oxidized LDL and accumulate their lipid. After a while they should differentiate into the anti-inflammatory M2 type which consumes the accumulated lipids through fatty acid oxidation for energy. This has nothing to do with creating disease but are all part of resolving the inflammatory situation. It is the healing process.

However, although the elements are there to perform this change from M1 towards M2, this process becomes only partially successful resulting in macrophages that fall in between showing properties of both M1 and M2. As a result they maintain M1 features including the uptake of oxidized LDL. The environment signals them in both directions.

My own interpretation of the research lead me to believe that the inflammation as a consequence of the hypoxia is not resolved by the actions of the macrophages although this is what they are supposed to do (as you can see in muscle repair). They normally resolve the hypxia-induced inflammation by resolving the hypoxia through cell regeneration and vascularisation.

If you have read my article on the root cause then you see that the cause is outside of the reach for macrophages to resolve it.

Total Picture

Due to the existence of a hypoxic region at the bifurcation as a result from contracted or hardened cell wall due to factors such as smoking, high insulinemia, fructose etc… You get a region in the blood flow that does not deliver sufficient oxygen to the deepest layer of the intima.

The furthest away from oxygen delivery becomes the most hypoxic and starts to send out inflammatory markers.

Monocytes infiltrate and are immobilized by hypoxia and differentiate into M1 macrophages by the environmental stimulus. As M1 macrophages they take up oxidized LDL so that they can collect lipids to support their proliferation. Normally the further progression of actions from M1 macrophages result in a morphology towards M2 macrophages.

These M2 macrophages use the accumulated fatty acids for fuel resulting in a clearance of the fatty acid content. We see that there is vascularisation penetrating from the vasa vasorum so the initial hypoxic region closest to the media is rescued from that side.

But at the same time the original M1 macrophages also stimulated growth of the intima creating more distance between the hypoxic region and the endothelial wall. On one hand the issue is resolved but on the other hand the problematic area now just shifted further away from the media and still the problem of the reduced blood flow isn’t solved.

Because this issue with the blood flow remains, you get a continuous growth stimulation leading to a further narrowing of the blood passage.

And now for the research that led me to this conclusion.

Where I started

A first thing I did was get me some better background info on macrophages. I know they are part of the immune system so a refresher is always in place. An excellent video to start with for a basic overview is the following.

Important to understand is that, as you can see from the video above, these cells develop and morph into different types. Macrophages circulate as monocytes and when triggered they develop into different types of macrophages.

Next up is a visit to wikipedia on macrophages itself. It has a section on macrophage subtypes but underneath I noticed a section on muscle regeneration. That is interesting. In my root cause research, vascular smooth muscle cells are affected. I know these cells are not exactly the same as skeletal muscle cells but still.. why not have a look at the interplay between skeletal muscle and macrophages. Maybe we can learn something from it.

The next video shows how muscle damage and repair is supported by monocytes-turned-macrophages.

What we can learn here is that macrophages play an active role in the environment and again note how macrophages of the M1 type transition into the M2 type. These are the 2 main categories that I’ll look into but note that there are many sub-categories and versions in between depending on the tissue they are located at etc…

By taking up malfunctioning cells macrophages have learned to extract the lipids for further usage. Related to this, a distinct feature is that M1 types are more prone to fatty acid synthesis while the M2 favor fatty acid oxidation. They operate in a hypoxic area so they revert to glycolysis, something we recognize from cancers cells that also live in hypoxic conditions.

“Macrophages and lipid metabolism” https://www.sciencedirect.com/science/article/pii/S0008874918300327

This is offering a first clue as to why M1’s take up lipids. Proliferating cells require lipids to build these new cells. Proliferating cells require lipids for membrane construction and other organelles that also have membranes.

M1

When the monocytes migrate into the inflammed area, hypoxia is one of the drivers to change their phenotype towards M1 macrophages.

“Hypoxia-Inducible Factor-1α Expression in Macrophages Promotes Development of Atherosclerosis” https://pubmed.ncbi.nlm.nih.gov/27444197

These type of macrophages stimulate inflammation in order to trigger proliferation of satellite cells which then further become differentiated and form newly repaired tissue.

These macrophages are also know to stimulate proteoglycans (PGs) to stimulate the buildup of the extracellular matrix which will hold the new tissue. These proteoglycans are the earliest evidence seen of atherosclerosis before lipids accumulate.

Walton [8] showed this mucoid thickening of the intima occurs before lipid infiltration and is composed primarily of collagen, PGs, and ECM. Thus, although lipid accumulation in the artery wall is considered an early event in atherosclerosis, lipid retention is not the initiating event, and the fatty streak is not the first sign of atherosclerotic injury [11,12].

source: https://www.ncbi.nlm.nih.gov/books/NBK2029/

“Monocyte to Macrophage Differentiation: Synthesis and Secretion of a Complex Extracellular Matrix” https://pubmed.ncbi.nlm.nih.gov/22351750/

It tells us that these macrophages arrive to the site of injury and then start to accumulate lipids. This order of sequence is important as some believe LDL gets stuck, oxidizes (or not) and cause inflammation and then macrophages arrive to resolve this.

The correct order is that inflammation attracts macrophages and then they start to accumulate lipids.

This alone is a reason to question if lowering LDL is a meaningful thing to do.

M2

These macrophages are the ones who help the proliferated satellite cells to differentiate and form restored tissue.

One of the contributing factors to make the switch from M1 to M2 is the activation through PPAR-γ and IL-4. PPAR-γ is activated by fatty acids so to no surprise, the M1 needs to accumulate fatty acids in order to transition.

“New Insights on the Role of Lipid Metabolism in the Metabolic Reprogramming of Macrophages” https://www.frontiersin.org/articles/10.3389/fimmu.2019.02993/full

It is a complex world, the IL-4 cytokines come from Th2, activating PPAR-γ so these T-cells also have a role to play in the process.

“PPARγ Activation Primes Human Monocytes into Alternative M2 Macrophages with Anti-inflammatory Properties” https://www.cell.com/cell-metabolism/references/S1550-4131(07)00166-0

“The Hunt for the Source of Primary Interleukin-4: How We Discovered That Natural Killer T Cells and Basophils Determine T Helper Type 2 Cell Differentiation In Vivo” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5924770/

Uptake of LDL

There are different scavenger receptors on the cell surface that take up fatty acids from circulation. LOX-1 and CD36 are 2 types of such scavenger receptors. CD36s are particularly fond of oxidized LDL (oxLDL). Just as they learned to extract fatty acids from dying cells, they also extract the fatty acids from oxLDL. Under normal circumstances, the cholesterol is expelled from the cell and picked up by HDL particles via a process called reverse cholesterol transport.

With such an important function for HDL it should not be a surprise that low HDL is stated as being a strong and independent risk factor for atherosclerosis! But does increasing HDL lower your risk? Attempts have been made and failed to show benefit. Is that a surprise though? Do you expect a fix if the root cause is not addressed?

“HDL-cholesterol and cardiovascular disease: rethinking our approach” https://pubmed.ncbi.nlm.nih.gov/26192490/

Another issue is the hypoxia itself, this needs to be resolved for the cholesterol export to function properly.

“Hypoxia-Inducible Factor-1α Expression in Macrophages Promotes Development of Atherosclerosis” https://pubmed.ncbi.nlm.nih.gov/27444197/

Interesting to note is that the availability of cholesterol within the cell triggers the transcription to export the cholesterol.

Accumulation of cellular cholesterol leads to activation of several transcription factors, including PPARγ, LXRs and RXRs which subsequently regulate expression of their target genes including transporters such as ABCA1 and ABCG1 which regulate the efflux of free cholesterol and scavenger receptors. Alternatively, passive efflux of free cholesterol can also occur.

source: https://www.sciencedirect.com/science/article/pii/S0008874918300327

So the idea is to export cholesterol yet we do find cholesterol back in the plaque so it seems that yet again the unresolved hypoxia is playing a disturbing factor.

But back to the oxLDL…

Oxidative stress

In areas of low oxygen you get an increase in ROS production which interacts with the LDL to oxidize it. Being in an environment with high risk of oxLDL, macrophages prefer oxLDL. That is not a coincidence.

oxidized LDL has also been reported to induce the expression of the M2 macrophage phenotypic marker arginase 1 via activation of peroxisome proliferator activated receptor-γ (PPARγ)⁷⁹, and oxidized phospholipids present in oxidized LDL induce a macrophage phenotype distinct from M1 or M2 that has been termed Mox, which is characterized by increased expression of nuclear respiratory factor 2 (NRF2)-dependent genes and reactive oxygen species⁸⁰. It is likely that T helper 1 (Th1) and Th2 cells in plaques secrete macrophage-polarizing factors⁸¹ that also contribute to the balance of M1 and M2 macrophages. Nonetheless, the factors in the plaque microenvironment that promote the polarization of these cells in vivo remain incompletely defined.

source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4357520/

This is a difficult area to conclude from because of its complexity but we can already see that oxLDL is a factor that contributes to the change away from the pro-inflammatory M1 type. Rather than considering oxLDL an issue, it may even be a necessary factor in the path towards tissue repair and resolution of the inflammation. But why aren’t they moving towards M2?

Why don’t they change to M2?

In order to have a possible answer I first looked at some info again on how this is handled in muscle tissue.

We see here that BACH1 is an inhibiting component for the transcription to move towards the M2 (repair macrophage) type. BACH1 gets inhibited by binding to heme.

Heme itself gets degraded by Heme Oxygenase 1. This enzyme gets upregulated by oxLDL but also by hypoxia and we see this in the plaque.

HO-1 is transcriptionally upregulated as a sensitive antiinflammatory protein by various types of oxidative stress, such as oxidized LDL,¹⁴ ultra-violet radiation,¹⁵ thiol scavengers,¹⁶ and hypoxia,^17,18 as well as substrate heme¹⁹ in the cardiovascular system.

source: https://www.ahajournals.org/doi/10.1161/01.atv.0000178169.95781.49

There are likely other factors but this again makes an important link with hypoxia. If the status of hypoxia cannot be resolved then this creates a chronic situation of more prevalent M1 macrophage phenotypical lipid uptake.

But the factor of oxidized LDL is also intriguing so I took a closer look at the reference. It seems there are specific molecules required in the oxLDL to trigger HO-1. This may actually represent an issue in the research as there are many ways to create oxLDL. Preferrably the test is done with oxLDL extracted from human sera.

Lysophosphatidylcholine, one of the major components present in oxidized LDL, was ineffective to induce the gene expression, suggesting that other lipophilic substances derived from LDL oxidation are responsible for the induction of HO-1

source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1858397/

Why don’t macrophages emigrate away?

A reason why macrophages stay where they are is because of hypoxia but also cholesterol accumulation induces expression of netrin 1, a molecule that prevents them to migrate. This is very important because as I showed in my previous article, there may be a chronic situation of hypoxia that initiates the rest of the pathology.

Macrophage expression of these migration inhibitory molecules are also induced during hypoxia, which is intimately linked to atherosclerosis^26,95

source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4357520/

In the process of resolving the situation there is vascularization, small micro vessels that are formed to feed the affected area so that it can receive nutrients and oxygen. This is how muscle develop and become stronger.

What would happen if that situation of hypoxia cannot get resolved?

LMHR

Some people on a ketogenic diet see their LDL cholesterol increase dramatically (typified Lean Mass Hyper Responder or LMHR). Levels above 300mg/dL is not unusual but typically they also see their HDL double compared to the days they were on a high carb diet.

Naturally such people are severely encouraged by their doctor to either quit the diet or use statins to lower the cholesterol because current assumption is that it causes atherosclerosis.

One of the elements in the physiology is that this CD36 receptor is enhanced by skeletal muscle. This means that if oxidized LDL is part of the issue, more of it is taken up by the skeletal muscle for energy usage.

oxLDL

OxLDL is no stranger to the body so is it really just bad? With my current understanding I suspect that the body has foreseen a way to make it part of the overall functioning. The oxidized form may no longer be able to perform the same functionality as LDL so rather than throwing it away, cells have developed a way to use the fatty acids that it contains.

This would be ideal and could explain the preference of the CD36 receptor for oxLDL. Recuperate what is possible. They contain energy which helps survival. Being on a ketogenic diet you especially depend on fatty acids for energy production so our LMHR’s could be clearing more of these oxLDL from the circulation leading to an overall lower level.

But we can only claim this if there is no proportional increase in oxLDL as a percentage of total LDL.

“Muscle-specific overexpression of FAT/CD36 enhances fatty acid oxidation by contracting muscle, reduces plasma triglycerides and fatty acids, and increases plasma glucose and insulin” https://pubmed.ncbi.nlm.nih.gov/10480880/

And again, such affinity for oxLDL by the CD36 scavenger has a reason so oxLDL must be part of the design. It cannot be bad by itself but perhaps in the quantities that it is present, it may overwhelm the system.

expression of the fatty acid translocase Cd36 gene was higher in the LCHF group (mouse study)

source: https://link.springer.com/article/10.1186/s12986-016-0132-8

Previously, we reported that FoxO1 activation in C₂C₁₂ muscle cells recruits the fatty acid translocase CD36 to the plasma membrane and increases fatty acid uptake and oxidation. This, together with FoxO1 induction of lipoprotein lipase, would promote the reliance on fatty acid utilization characteristic of the fasted muscle.

source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2386936/

It makes me suspect that absolute levels of oxLDL are more important than your LDL-c level.

The following article even shows that the LDL receptor is downregulated (!) in macrophages so that non-oxidized LDL have a more difficult time being absorbed while the scavenger receptors are not downregulated.

among 425 patients with acute coronary syndrome (ACS), the HR of AMI recurrence or ACS-related death at the 5-year follow-up was 2.88 (95% CI: 1.93–4.32) for 1 mmol/L increase in circulating ox-LDL

“Association between circulating oxidized low-density lipoprotein and atherosclerotic cardiovascular disease” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627698/

When a hazard ratio goes above 2 then it gets me interested for a possible link. The article discusses other studies that showed no meaningful HR so we have to be careful here and look at each study to see what differs between them for reaching different conclusions. It could also show that oxLDL is just one factor of many that make up the pathology of atherosclerosis.

The article continues to show that different statins are able to lower the volume of oxLDL. So if statins exert an effect, is it because they lower LDL cholesterol overall or because they lower oxLDL?

A different paper reports on the ability of Atorvastatin to suppress the uptake of oxLDL by macrophages and at the same time lowers their CD36 expression.

“Atorvastatin therapy in hypercholesterolemic patients suppresses cellular uptake of oxidized-LDL by differentiating monocytes” https://pubmed.ncbi.nlm.nih.gov/12119208/

Whatever the answer, it doesn’t change the observed mechanism of reduced uptake of normal LDL. And with how statins exert their effect, should we consider our total LDL-c level as an issue?

I’m more inclined to focus on oxLDL. Not to eliminate it but to reduce quantity.

HDL

Also when we see the role that HDL has to play, we can conclude it is not necessarily the cholesterol itself that can form an issue. LMHR’s more often than not have very high HDL cholesterol to assist in this uptake from cells that do not need the cholesterol such as the macrophages. But since all cells that have increased their dependency on fat metabolism have increased uptake of oxLDL, there could simple be more HDL required.

Is the level in function of how much is needed? We can’t be too quick to conclude. They don’t necessarily have a higher availability for cholesterol uptake by HDL from macrophages.

This is a very difficult area of research I think because it will all come down to the rate at which this happens and the amount of oxLDL that is in circulation.

“Macrophage‐mediated cholesterol handling in atherosclerosis – Formation of cholesterol esters” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4717859/#jcmm12689-sec-0007title

Furthermore the cholesterol efflux speed depends on the ABC1 transporter which is down regulated by unsaturated fat, not saturated fat. The ketogenic diet favors more on the side of saturated or mono-unsaturated fat and certainly promotes avoiding poly-unsaturated fat.

“Unsaturated fatty acids repress expression of ATP binding cassette transporter A1 and G1 in RAW 264.7 macrophages” https://pubmed.ncbi.nlm.nih.gov/22209005/

In addition, do not forget that HDL also has an anti-oxidant function for LDL.

Finally

What would I recommend knowing what I know so far? Handle the root cause and don’t worry about cholesterol but do look into what causes LDL to oxidize. Check out my article on oxidized LDL for some of the protective factors. You can find them more towards the end.

I hope this article has given you again some more background on how things work and help you in your search to be in control of your health.

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