June 19, 2023
More on HDL Biology from the CETP side or Cholesterol Ester Transfer Protein
This is exciting stuff!
High Density Lipoprotein -
Let us head back to the headwaters of HDL biology to find some more answers.
New information: An amazing podcast with Peter Attia and John Kastelein has given us some new stuff to chew on. Again we go deep here as this information is linked to the leading cause of death worldwide - heart disease. I had to pull a bunch of these papers to fully understand this information and it was worth it.
If you did not read the original HDL piece or remember the basics of HDL biology, I have attached the original piece below the bold underlined sentence below.
HDL as an associated biomarker of death risk has a U shaped curve with higher all cause mortality at very low and high levels of volume. Let us understand why? (Madsen et. al. 2017)
Anything that causes more LDL, low density lipoproteins, to stay in circulation will raise one's risk of ASCVD or heart attack. The historical reality (as I have discussed for years) for why we would have these genomic mutations to have more LDL particles in circulation is 1) as a protection mechanism against bacterial infections which were common for thousands of years. The HDL and LDL particles have receptors on their surface to grab bacterial cell wall debris like LTE or LPS and remove them via the liver. This is a massive beneftit to the human species until recent times. 2) as a storage mechanism for calories/recirculation of metabolically expensive cholesterol. (Maile et. al. 2020)(Feng et. al.. 2019)(Trinder et. al. 2021)
The kicker here is disease associations. If you are living in 5000 BC, the elevated LDL particle number was protective because in general no one had diabetes or other chronic disease of aging that would hijack immune function. The LDL particle and HDL particles could grab bacterial endotoxin and clear it via the liver reducing immune stimulation and death risk. Now, when studied, ICU data shows that pre illness elevated LDL is protective against bacterial sepsis death unless you have comorbid immune dysfunctional diseases like diabetes, gout, hypertension. (Feng et. al. 2019) This is so similar to the data of the Tsimane Indians of South America who have elevated c reactive protein and LDL levels in the absence of chronic diseases of aging. They are protected against bacterial disease with their elevated lipoproteins and don't suffer our diseases because of the lack of exposure to our foods, toxins and sloth that drive dysfunctional lipid and immune biology.
Back to the HDL story. There is a series of new data sets coming out that seem to point to HDL biology being beneficial to human health if the CETP or cholesterol ester transfer protein is mutated or non functional. Genome wide association studies clearly show that CETP is a longevity gene when it is hypo functioning. I.e it is not transferring cholesterol molecules from HDL to LDL particles for transport and instead keeping the cholesterol.
Let us discuss CETP.
CETP is a gene that encodes for a protein to be made in the liver with main functions of: 1) facilitating transfer of both cholesteryl esters from high-density lipoprotein (HDL) particles to apolipoprotein B containing LDL/VLDL and other particles 2) facilitating the transfer of triglycerides from apoB particles to HDL particles. These effects are all about energy transfer and cholesterol recycling in a resource scarce environment.
Again, loss of function mutations in the CETP gene encodes for a longevity gene that keeps ApoB (LDL/VLDL) protein levels low and is associated with significantly reduced ASCVD risk reductions. (Nurmohamed et.. al. 2022)
We have to look at this CETP protein in the context of human evolution. From a recent paper: "Modern humans are effectively stone-agers living in a world vastly different from that of our ancient past, which involved food insecurity and countless other hardships. Routine seasonal food shortages can constrain human reproduction, and volcanic winters and other episodic catastrophes in our past caused sustained, widespread famines that may have bottlenecked our global population to just a few thousand breeding pairs. Those who survived and reproduced under such harsh conditions may have been more likely to have so-called ‘thrifty’ genotypes that made them more efficient at acquiring, utilizing, and storing calories and other nutrients. But around 10 000–20 000 years ago, humankind started gaining relief from several major burdens that limited our survival and reproduction, including starvation, predation, and infectious disease. These and other changes stabilized the human genome much as it was 10,000 years ago, with thrifty and non-thrifty genotypes alike now enjoying a remarkably higher probability of reproducing compared with just 2000 years ago. But when confronted with modernized societies’ high-calorie diet and limited physical activity, those with thrifty genotypes may be predisposed to ‘afflictions of affluence’, which may include diabetes, obesity, and hypercholesterolaemia. In theory, traits predisposing us to hypercholesterolaemia could have evolved to ensure adequate cholesterol levels. However, the biology of cholesterol synthesis and cholesterol and triglyceride (TG) transport, as described below, suggest that those traits evolved to ensure adequate energy and energy stores." (Laufs et. al. 2019) I WOULD ENCOURAGE EVERYONE TO READ THIS PAPER.
Cholesterol is very metabolically expensive to make utilizing 36 ATP in production. Thus, the human body would not want to waste it historically. Transferring it to different tissues or back to the liver via HDL to LDL transfer would be preferable in a time where resources were scarce leading to recirculation and reuse via the liver and elevations in the blood stream during periods of food excess. Also, triglycerides (TG) are our storage form of fat as energy. Normally, we transfer TG from VLDL and LDL particles to HDL in exchange for cholesterol using CETP. In periods of food scarcity, sending the triglycerides to the HDL particles would facilitate more energy deposition in adipose tissue when food was found to be used during food scarcity later on. This is also a net benefit for reproduction as fat stores are critical for females to procreate. So, we could assume that CETP was very very useful as an energy storage protein in the past. (Now it is a problem - not then) APOE4 geneotypes also appear to follow this same line of thinking - more on that another day when we look at neurodegenerative disease.
Fast forward to Dr. Kastelein's work. CETP inhibitor drugs that just raise HDL levels have all been failures in lowering ASCVD risk. However, newer work by Dr. Kastelein has shown that if the CETP inhibitor is raising HDL, lowering LDL and improving function, then it will have just shy of 50% risk reduction. This is a significant risk reduction. (Nurmohamed et.. al. 2022) What we are learning through evolution and new science is that if the drug can block the transfer of TG to the HDL and cholesterol to the LDL, then the risk of an LDL gradient occurring drops because the absolute number of particles with ApoB attached goes down leading to less lipoprotein finding its way into the heart vessel wall. His new data also shows that with the correct CETP inhibition, the liver will make more ApoA1 which in turn makes more HDL particles to grab peripheral cholesterol wherever it is in excess reducing cardiac event risk.
Dr. Trinder and colleagues have published a nice paper looking at CETP inhibition and HDL levels in relation to bacterial infection and sepsis risk. From Dr. von Eckardstein's paper: "HDLs bind potentially toxic substances, such as bacterial lipopolysaccharides, oxidized lipids, as well as some lipophilic xenobiotics. In plasma, potentially hazardous molecules are either eliminated by reverse transport to the liver or inactivated directly on the surface of HDLs. The best-investigated example for the latter situation is the hydrolysis of oxidized phospholipids by paraoxonase 1, lipoprotein-associated phospholipase A2, and LCAT. HDLs also exert direct antimicrobial effects on viruses and even protozoa. At least in vitro, HDL or apoA-I interfere with the entry or fusion of viruses with target cells. Of note, SR-BI is an entry route of several viruses, including SARS-CoV-2, into cells and this process may be competed by HDLs. Finally, the proteome of HDLs is enriched with proteases and protease inhibitors which modulate platelet aggregation, coagulation, fibrinolysis, complement activation, and tissue degradation. They help to counteract downstream adverse effects of injuries, infections, and inflammation and support wound healing. Of note, functionally related proteins tend to cluster within distinct sub-populations of HDL." (von Eckardstein et. al. 2023)
In other words, when HDL is functioning normally it has effects far beyond simple cholesterol triglyceride reverse transport, but actually is a major player in cell signaling and immune activity. The clearance of bacterial cell wall debris from the blood stream is so important to avoidance of death during a serious bacterial infection. The removal of oxidized fats from the peripheral tissues and blood is critical to the avoidance of inflammation that destroys local tissue..... so much more to learn he about the pleotrophic actions of the HDL lipoprotein.
Here comes the RUB! HDLs can lose these protective functions in our modern world leading to worsened chronic diseases. The key to the loss of function is still to be worked out but appears to be related to inflammation and our modern lifestyles. Autoimmunity and other inflammatory illnesses have dysfunctional HDL biology as a hallmark. (Feingold et. al. 2022)
Ultimately, it again comes down to controlling all of the upstream targets of inflammation in order to reduce the disease burden of ASCVD.
The article from a few weeks ago is recopied here for a refresher as this information is tricky for me and I have read it many times.
Review: What is the biology? HDL is the main source of reverse cholesterol transport (RCT) which is to say that HDL particles transfer excess cholesterol molecules from systemic cells, such as arterial wall immune based macrophages or foam cells, into the themselves whereby they are esterified to form cholesteryl esters (CE). Lecithin–cholesterol acyltransferase, LCAT, esterifies cholesterol to CE which is hydrophobic causing it to migrate from the HDL particle surface to the core. This action in effect removes cholesterol from peripheral cells and keeps them away which is thought to be a part of the benefit of HDL. The HDL particle then travels through the blood stream to the liver where the cholesterol as an ester molecule is taken up by the liver by a hepatic scavenger receptor called (SRB1) or other HDL receptors. The liver then recycles it and/or excretes it into the bile for discharge via the bile duct and ultimately in stool. On the other hand, HDL can transfer the cholesterol to an LDL particle increasing the volume of LDL particles which are the major drivers of CVD in humans. A major feature of CVD is the deposition of small dense LDL particles in the heart artery vessel wall. When a macrophage, immune cell that engulfs pathogenic molecules, swallows a small dense oxidized LDL, it begins an inflammatory response that leads to a metabolically and immunologically active cell called a foam cell. Oxidation is a process that occurs whereby oxygen radicals released by immune cells attack the LDL particles in the vessel wall leaving them damaged by oxidation and furthering a loop effect more immune activation. HDL particles can prevent this process by releasing certain enzymes that block the oxidation response. This maybe another method of cardio-protection by the HDL molecule.
HDL lipoproteins carry a surface protein called apolipoprotein A1, Apo A1, which is involved in cell signaling leading to the RCT and antioxidant effects of HDL. Interestingly, systemic inflammation reduces the action and volume of Apo A1 protein. Inflammation in the body can raise the levels of acute phase reactants like ferritin, c reactive protein and amyloid which can cause the Apo A1 protein to not function properly leaving the RCT and anti inflammatory effects of HDL dysfunctional.
Therefore, anything that causes systemic inflammation can negatively affect HDL activity worsening CVD by blocking reverse cholesterol transport leaving more oxidized LDL in the heart vessel wall. As we have noted for years in this newsletter, the upstream causes of systemic inflammation are related to nutrition, mental/physical stress, sleep, toxin load, sloth and more. (hypothesis) - It is no wonder that HDL pharmaco-therapies are failures since the upstream targets that render HDL inactive in RCT and/or oxidation are untouched by HDL enhancing medical therapy. (Rader D. 2014)
Other recently discovered desired effects of the HDL particle are: a) the induction of endothelial nitric oxide synthase, eNOS, which is an enzyme that produces nitric oxide relaxing blood vessels, b) the enhancement of insulin secretion and glucose metabolism, c) increased activity of adenosine monophosphate kinase, AMPk, d) increase lipoprotein lipase which helps to metabolize triglycerides and fats, e) enhance immune activity. (Bardagjy et. al. 2019) These effects are all pro health and anti-inflammatory in nature causing a loop effect that enhances HDL function and reducing CVD risk.
What can we learn from disorders of HDL function or volume? There is an inherited rare disease called Tangier disease where the RCT of HDL volume is so low that the activity is minimal leaving affected individuals with cholesterol ester filled macrophages throughout the body, like foam cells of the heart like CVD, orange colored tonsils or enlarged liver and spleen. Other diseases like : Familial HDL deficiency is associated with very low serum HDL concentrations and premature CHD; LCAT deficiency is due to Lecithin cholesterol acyltransferase deficiency. presents with annular corneal opacity, and progressive renal disease with proteinuria. Other causes that make a low HDL including taking Drugs such as beta-blockers, benzodiazepines, and anabolic steroids. (Alshaikhli et. al. 2022) Mendelian randomization studies have failed to show a strong causal link between HDL number and premature CVD risk.
These diseases show in principle that HDL is important to the progression of CVD, however, this is likely through inflammation and infection reduction and not directly through RCT. The question remains, how and through what mechanisms? More data is needed to answer this question as we are clear that raising HDL number is not changing CVD risk.
Let us switch gears and think outside the box here as we did with the LDL cholesterol discussion. Why would we have evolved with HDL molecules other than energy and cholesterol movement? The answer as it was with LDL particles belongs in the immune sphere of influence. From the Journal Cardiovascular Research: “During infections or acute conditions high-density lipoprotein levels decrease very rapidly and HDL particles undergo profound changes in their composition and function. These changes are associated with poor prognosis following endotoxemia or sepsis and data from genetically modified animal models support a protective role for HDL. The same is true for some parasitic infections, where the key player appears to be a specific and minor component of HDL, namely apoL-1. The ability of HDL to influence cholesterol availability in lipid rafts in immune cells results in the modulation of toll-like receptors, MHC-II complex, as well as B- and T-cell receptors, while specific molecules shuttled by HDL such as sphingosine-1-phosphate (S1P) contribute to immune cells trafficking. Animal models with defects associated with HDL metabolism and/or influencing cell cholesterol efflux present features related to immune disorders. All these functions point to HDL as a platform integrating innate and adaptive immunity.” (Catapano et. al. 2014)
A critical role of HDL particles, like LDL, is the natural innate ability to grab bacterial cell wall debris in circulation, bind it and clear it via the liver. This action reduces immune activation locally and systemic inflammation in total. Bacteria have lipid outer membranes like lipopolysaccharide, (LPS), of Gram-negative bacteria, or lipoteichoic acid, (LTA), of most Gram-positive bacteria. These cell wall membrane pieces are highly inflammatory if found in circulation. We know this to be true based on animal models with reduced HDL activity leading to increased inflammation in response to a bacterial infection.
As discussed with LDL and innate immunity, reduced HDL lipoprotein levels pre-infection increases the overall risk of sepsis post infection. If HDL levels are high pre-infection, then they will drop rapidly as they clear the bacterial cell wall debris leading to a better outcome overall. Although the absolute number matters, I think that the function or lack there of for the HDL particles likely matters more.
HDL volume has been highly associated with inflammation, immune activation and disease in study. HDL knockout mice models have shown enlarged peripheral lymph nodes and spleen with increases in immune cells of the T and B cell lineage and macrophages. As note earlier in the low HDL disease Tangiers, these immune cells have an altered function and the lack of HDL molecules leads to an inability to remove cholesteryl esters from macrophages leaving them dysfunctionally prone to stimulating inflammation. Over time these problems can develop an autoimmune or inflammatory immune polarity. The abnormal immune activity can lead to problems with infection as there is a direct effect of the action of these Apo A1 particles and immune activation in a necessary response to bacterial, parasitic and viral infections. (Bonacina et. al. 2021)
Many autoimmune diseases like rheumatoid arthritis, Crohn’s disease and Systemic Lupus Erythematosus are are associated with a significantly higher risk of CVD and are noted to have HDL particles that are low in absolute number, but also have a characteristic dyslipidemia with high triglycerides (TG), high LDL with the low HDL particle number. The macrophages and white blood cells conversely have high volumes of cholesterol esters inside them rendering them prone to innate immune activation and inflammation. The abnormal HDL particles are enriched with TG and depleted in CE resulting in attenuated anti-oxidative activity, reduced anti-inflammatory effects, and lower capacity to promote cholesterol efflux.Therefore, as the data emerges, it appears that the function is key to health outcome although absolute number matters in the case of infection or sepsis. In autoimmune conditions, HDL functional activity is broken. The composition of the HDL cell membrane and thus it’s activity appears to be pro inflammation and pro autoimmune disease.
Look at this statement by Dr. Catapano. "Also patients with type 2 diabetes mellitus, a clinical condition recently redefined as an autoimmune disease rather than just a metabolic disorder, exhibit an impaired HDL metabolism and the presence of dysfunctional HDL. Whether the immune-related activities of HDL have a role also in this context beyond the known metabolic functions is an intriguing hypothesis, which is so far supported by circumstantial observations." (Catapano et. al. 2014)
Remember that ApoA-I is the main structural and functional apoprotein of the lipoprotein HDL and plays a key role in the induction of cholesterol removal from peripheral cells with the plan to take them back to the liver for clearance. This interaction with cells results in cholesterol depletion and disruption of intracellular signaling in the cell membrane lipid rafts. These lipid rafts allow for receptors with key immunological functions like toll-like receptors (TLRs) and T- and B-cell receptors (TCR and BCR) to be active and immune stabilizing. We now know that the lipid composition of rafts determines their function and that any modification of lipid raft composition can modulate signaling altering immune cell biological functions. (Catapano et. al. 2014) More data is emerging that the HDL molecule will deplete cholesterol from the lipid rafts altering cell signals to be anti-inflammatory and pro resolution of systemic inflammation. When infusing HDL into an arthritic mouse with bacterial inflammation, the response was to reduce all inflammation via the downregulation of Toll like receptors and immune cell signaling. I assume that these HDL cells were fully functional. This may be the key to the failed HDL pharma trials. If the drug was increasing a person's HDL number, but not improving function, ..... failure.
When looking at the diverse immune actions of APO A1 HDL cells we see so many that are involved in immune homeostasis and reduced inflammation! Decreased Toll like receptor signaling; white blood cell activation and proliferation; dendritic cell differentiation, maturation, and antigen presentation; interferon response; macrophage activity; M1 macrophage polarity. For the non medical professionals, this is the key: These changes are ALL associated with reduced inflammation, autoimmunity and disease in general.
Covid death was highly associated with dyslipidemia and especially a low HDL level. "Low and high LDL-C, low HDL-C, and high TG levels were negatively associated with COVID-19-related mortality." (Aydin et. al. 2022) Coincidence? I think not! These dyslipidemic changes are all a sign a broken lipoprotein functionality and this leads to poor immune infection action, clearance and resolution.
There was a reason all along for the generation of abnormally high or low volumes of lipoproteins in human history. That reason was survival from infection. As always, the genetics of CVD are less about a mistake of humanity’s evolution and more a mistake of our modern lifestyle in relation to our evolving genetics.
The biggest and most profound understanding for me after doing this deep dive into LDL and HDL biology in humans is the understanding that our lifestyle choices are driving dyslipidemia altering immune lipid functional capacity leaving us in a pro inflammatory state and prone to disease of aging including autoimmunity, CVD and more.
What is the answer? Everything discussed in last weeks newsletter! See more in section III below.
Dr. M
von Eckardstein Europ Heart Journal
Feingold Endotext
Catapano Cardiovascular Research
Peltonen Human Molecular Genetics
From Cells: A powerful tool to appreciate the role of HDL comes from studies on monogenic disorders resulting in extremely low or high HDL-C levels. Monogenic disorders associated with low HDL-C levels include those related to loss-of-function mutations in APOA1, the gene encoding apoA-I, LCAT, encoding lecithin-cholesterol acyl transferase (LCAT), and ABCA1, encoding ABCA1. Genes implicated in monogenic disorders associated with high HDL-C levels include CETP, encoding cholesteryl ester transfer protein (CETP), SCARB1, encoding scavenger receptor class B member 1 (SRB1), and LIPC, encoding hepatic triacylglycerol lipase [142]. Patients with Tangier disease, despite having very low HDL-C levels due to ABCA1 deficiency, do not manifest premature coronary artery disease, but rather present an increased inflammatory status [143]; this has been associated to an increased inflammasome activation triggered by reduced cholesterol efflux from myeloid cells [144]. Moreover, mendelian randomization studies failed to demonstrate a causal link between HDL-C and CVD risk [145], but rather indicate that low HDL-C levels increase the risk of infections [6]. In addition, elevated peripheral blood leukocyte counts were reported in subjects with low HDL-C levels of any origin [146]. In line with this observation, subjects carrying a low HDL-C polygenic score have been shown to present an increased risk of hospitalization for infections [147]; this is consistent with the increased mortality for sepsis reported in subjects with gain-of-function mutation on CETP, who present a dramatic reduction of HDL-C levels during infection [148]. It is tempting to speculate that CETP, beyond transferring cholesteryl esters from HDL to apolipoprotein (apo) B-containing triglyceride-rich lipoproteins (TRLs), could also participate to immune response following a bacterial insult. This hypothesis is supported by the evidence that CETP is predominantly produced by Kupffer cells, specialized liver macrophages, and that its expression is reduced during inflammation; in turn, this leads to an increase in HDL-C levels that may contribute to LPS clearance during infections [149]. Similarly, LCAT deficiency in mice was associated with the presence of immature discoidal HDL and a reduced LPS-neutralizing capacity [150], thus suggesting that not only the concentration but also the type of HDL subclasses differently modulates immune responses [151] (Bonacina et. al. 2021)
Dr. M
