A Hunt for Causes of Atherosclerosis

Using the most advanced tools of molecular biology to sift through the sands of the genome for a little gold.

Blood vessels have a hard life. Every time you put on shoes, the vessels in your feet get smashed and smooshed, for hours on end. And do they complain? Generally, not much. They bounce back and make do with the room you give them. All through the body, vessels are subject to the pumping of the heart, and variations in blood volume brought on by our salt balance. They have to move when we do, and deal with it whenever we sit or lie on them. Curiously, it is the veins in our legs and calves, that are least likely to be crushed in daily life, that accumulate valve problems and go varicose. Atherosclerosis is another, much more serious problem in larger vessels, also brought on by age and injury, where injury and inflammation of the lining endothelial cells can lead to thickening, lipid/cholesterol accumulation, necrosis, calcification, and then flow restriction and fragmentation risk. 

Cross-section of a sclerotic blood vessel. LP stands for lipid pool, while the box shows necrotic and calcified bits of tissue.

The best-known risk factors for atherosclerosis are lipid-related, such as lack of liver re-capture of blood lipids, or lack of uptake around the body, keeping cholesterol and other lipid levels high in the blood. But genetic studies have found hundreds of areas of the genome with risk-conferring (or risk-reducing) variants, most of which are not related to lipid management. These genome-wide association studies (or GWAS) look for correlations between genetic markers and disease in large populations. So they pick up a lot of low-impact genetic variations that are difficult to study, due to their large number and low impact, which can often imply peripheral / indirect function. High-impact variations (mutations) tend to not survive in the population very long, but when found tend to be far more directly involved and informative.

A recent paper harnessed a variety of modern tools and methods to extract more from the poor information provided by GWAS. They come up with a fascinating tradeoff / link between atherosclerosis and cerebral cavernous malformation (CCM), which is distinct blood vessel syndrome that can also lead to rupture and death. The authors set up a program of analysis that was prodigious, and only possible with the latest tools. 

The first step was to select a cell line that could model the endothelial cells at issue. Then they loaded these cells with custom expression-reducing RNA regulators against each one of the ~1600 genes found in the neighborhood of the mutations uncovered by the GWAS analyses above, plus 600 control genes. Then they sequenced all the RNA messages from these single cells, each of which had received one of these "knock-down" RNA regulators. This involved a couple hundred thousand cells and billions of sequencing reads- no simple task! The point was to gather comprehensive data on what other genes were being affected by the genetic lesion found in the GWAS population, and then to (algorithmically) assemble them into coherent functional groups and pathways which could both identify which genes were actually being affected by the original mutations, and also connect them to the problems resulting in atherosclerosis.

Not to be outdone, they went on to harness the AlphaFold program to hunt for interactions among the proteins participating in some of the pathways they resolved through this vast pipeline, to confirm that the connections they found make sense.

They came up with about fifty different regulated molecular programs (or pathways), of which thirteen were endothelial cell specific. Things like angiogenesis, wound healing, flow response, cell migration, and osmoregulation came up, and are naturally of great relevance. Five of these latter programs were particularly strongly connected to coronary artery disease risk, and mostly concerned endothelial-specific programs of cell adhesion. Which makes sense, as the lack of strong adhesion contributes to injury and invasion by macrophages and other detritus from the blood, and adhesion among the endothelial cells plays a central role in their ability / desire to recover from injury, adjust to outside circumstances, reshape the vessel they are in, etc.

Genes near GWAS variations and found as regulators of other endothelial-related genes are mapped into a known pathway (a) of molecular signaling. The color code of changed expression refers to the effect that the marked gene had on other genes within the five most heavily disease-linked programs/pathways. The numbers refer to those programs, (8=angiogenesis and osmoregulation, 48=cell adhesion, 35=focal adhesion, related to cell adhesion, 39=basement membrane, related to cell polarity and adhesion, 47=angiogenesis, or growth of blood vessels). At bottom (c) is a layout of 41 regulated genes within the five disease-related programs, and how they are regulated by knockdown of the indicated genes on the X axis. Lastly, in d, some of these target genes have known effects on atherosclerosis or vascular barrier syndromes when mutated. And this appears to generally correlate with the regulatory effects of the highlighted pathway genes.

"Two regulators of this (CCM) pathway, CCM2 and TLNRD1, are each linked to a CAD (coronary artery disease) risk variant, regulate other CAD risk genes and affect atheroprotective processes in endothelial cells. ... Specifically, we show that knockdown of TLNRD1 or CCM2 mimics the effects of atheroprotective laminar blood flow, and that the poorly characterized gene TLNRD1 is a newly identified regulator in the CCM pathway."

On the other hand, excessive adhesiveness and angiogenesis can be a problem as well, as revealed by the reverse correlation they found with CCM syndrome. The interesting thing was that the gene CCM2 came up as one of strongest regulators of the five core programs associated with atherosclerosis risk mutations. As can be guessed from its name, it can harbor mutations that lead to CCM. CCM is a relatively rare syndrome (at least compared with coronary artery disease) of localized patches of malformed vessels in the brain, which are prone to rupture, which can be lethal. CCM2 is part of a protein complex, with KRIT1 and PDCD10, and part of a known pathway from fluid flow sensing receptors to transcription regulators (TFs) that turn on genes relevant to the endothelial cells. As shown in the diagram above, this pathway is full of genes that came up in this pathway analysis, from the atherosclerosis GWAS mutations. Note that there is a repression effect in the diagram above (a) between the CCM complex and the MAP kinase cascade that sends signals downstream, accounting for the color reversal at this stage of the diagram.

Not only did they find that this known set of three CCM gene are implicated in the atherosclerosis mutation results, but one of the genes they dug up through their pipeline, TLNRD1, turned out to be a fourth, hitherto unknown, member of the CCM complex, shown via the AlphaFold program to dock very neatly with the others. It is loss of function mutations of genes encoding this complex, which inhibits the expression of endothelial cell pro-cell adhesion and pro-angiogenesis sets of genes, that cause CCM, unleashing these angiogenesis genes to do too much. 

The logic of this pathway overall is that proper fluid flow at the cell surface, as expected in well-formed blood vessels, activates the pathway to the CCM complex, which then represses programs of new or corrective angiogenesis and cell adhesion- the tissue is OK as it is. Conversely, when turbulent flow is sensed, the CCM complex is turned down, and its target genes are turned up, activating repair, revision, and angiogenesis pathways that can presumably adjust the vessel shape to reduce turbulence, or simply strengthen it.

Under this model, malformations may occur during brain development when/where turbulent flow occurs, reducing CCM activation, which is abetted by mutations that help the CCM complex to fall apart, resulting (rarely) in run-away angiogenesis. The common variants dealt with in this paper, that decrease risk of cardiovascular disease / atherosclerosis, appear to have similar, but much weaker effects, promoting angiogenesis, including recovery from injury and adhesion between endothelial cells. In this way, they keep the endothelium tighter and more resistant to injury, invasion by macrophages, and all the downstream sequelae that result in atherosclerosis. Thus strong reduction of CCM gene function is dangerous in CCM syndrome, but more modest reductions are protective in atherosclerosis, setting up a sensitive evolutionary tradeoff that we are clearly still on the knife's edge of. I won't get into the nature of the causal mutations themselves, but they are likely to be diffuse and regulatory in the latter case.

Image of the CCM complex, which regulates response to blood flow, and whose mutations are relevant both to CCM and to atherosclerosis. The structures of TLNRD1 and the docking complex are provided by AlphaFold. 

This method is particularly powerful by being unbiased in its downstream gene and pattern finding, because it samples every expressed gene in the cell and automatically creates related pathways from this expression data, given the perturbations (knockdown of expression) of single target genes. It does not depend on using existing curated pathways and literature that would make it difficult to find new components of pathways. (Though in this case the "programs" it found align pretty closely with known pathways.) On the other hand, while these authors claim that this method is widely applicable, it is extremely arduous and costly, as evidenced by the contribution of 27 authors at top-flight institutions, an unusually large number in this field. So, for diseases and GWAS data sets that are highly significant, with plenty of funding, this may be a viable method of deeper analysis. Otherwise, it is beyond the means of a regular lab.

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The Politics of Martyrdom Invite the Politics of Extermination

Israel is following a kind of logic, but where does it lead?

We are all horrified by the war on Gaza. But there is some logic to it, despite the pervasive lack of reason. Hamas has set itself up as the never-say-die defenders of Palestinian ideals. Which appear to consist of the non-existence of Israel, and the resumption of Palestinian occupation of all its historical lands. (Though it has to be said that they had been ruled by a long succession of outside powers, from the Romans to the British.) Hamas cared little about the actual Palestinians, who have been pawns in its larger play for power and martyrdom. Like the PLO in earlier decades, Hamas has largely been carrying on an idealistic PR campaign of moral absolutism, resisting the occupier, to the last breath and drop of blood. So they posited that the survival of any member of Hamas was a victory for the "movement". This makes sense in a PR sense, as a moral position against oppression.

But all this also implies the nature of the Israeli response. Against an enemy that never negotiates, or concedes, or makes peace, the only solution (as the Romans found millennia ago) is extermination. Against an enemy that hides behind its own civilians and thinks nothing of operating out of hospitals and schools, in an attempt to play on the residual morality of its enemies, one can understand that compassion takes a back seat to effectiveness.

This is not to say that Israel is right either. They have compounded their original sin of terrorism and violent expropriation by more violence, more injustice, and the slow torturous drip of more expropriation. The West Bank continues to be eaten away by settlers and official Israeli state policy, and Gaza was under debilitating embargoes / sieges. Israel thought it was secure behind its border wall, but it turned out not to be so beautiful after all, for either side. But after the long historical process that has played out, Israel has all the power- the bombs, the planes, the organized political system and military. Hamas and the Palestinians generally have lost every war, and have little but their moral indignation and hatred to stand on. Turning that into extremist absolutism and martyrdom is a recipe for an even worse outcome. 

Their Muslim neighbors and allies took a couple of stabs at helping them, but lost each war to Israel, in comprehensive fashion. Their continuing assistance to the Palestinians is little more than cynical ploy to keep the Palestinians irritated and irritating, without risking their own necks. This was first the role of Saudi Arabia and the Gulf states, but that mantle has more recently been taken up by Iran. And now Israel is calling Iran's bluff, taking the fight to all its proxies and to Iran itself. They have not set themselves up for extermination, but rather for deterrence, whatever level of destruction that will take. The idea that Hezbollah could just pepper Israel with artillery at will without any reply was not realistic. 

So, can Israel carry out all these logical aims- to exterminate Hamas and degrade Hezbollah and Iran sufficiently to deter further guerilla warfare? That depends on its resolve and military capacity. But it also depends on its resolve to diffuse the overall Palestinian conundrum that, in the grand scheme of things sponsors all these antagonisms, by making a grand deal that finalizes a Palestinian state and territory. And that is where the current government of Israel is fatally uninterested and incapable. It looks like this government would rather be the new Rome, projecting power throughout the Middle East, collectively punishing Palestinians, and forcing Palestinians into a modern international diaspora, (and making itself into a semi-autocratic militarized state along the way), than it is in learning from its own history.

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Pumping DNA

Arnold has nothing on the DNA pumps that load phages.

DNA is a very unwieldy molecule. Elegant in concept, but as organisms accumulated more features and genes, it got extremely long and twisty. So a series of management proteins arose, such as helicases and gyrases to relieve the torsional tension, and topoisomerases to cut and pass strands through each other to resolve knots. Another class is DNA pumps, which can forcefully travel along DNA to thread it into useful spaces, like the head of a phage, or a domain in our nucleus, to facilitate transcriptional isolation or organized recombination and synapsis. While other motors, acting on actin and microtubules, manage DNA segregation during mitosis, cell division, and cell movement, true DNA motors deal directly with DNA.

An iconic electron micrograph of a phage with its head blown open. The previously enclosed DNA is splayed about, suggesting the capsid's great capacity for DNA, and great pressure it was under. Inset shows an intact phage. Note the landing tentacles, which attach to the target bacterium.

There are several types of DNA pump, the lower-powered of which I have reviewed previously. The champions in terms of force, however, are the pumps that fill phage heads. Phages are viruses that infect bacteria, and they operate under a variety of limitations. Size is one- they have to be small and have small genomes, due to the small size of their targets, the brevity of their life cycle, and the mathematics of scattered propagation. Bacterial cells are under turgor pressure, of about three atmospheres, and have strong cell walls to hold everything in. So their infecting phages have several barriers to overcome. One solution is to be under even higher pressure themselves, up to about sixty atmospheres. That way, once the injection system has cut through the cell wall and inner membrane, the phage genome, which is pretty much the only thing in the phage head (or capsid), can shoot out rapidly and take over the cell. 

Schematic of late phage development, where the motor (blue) docks to the phage head and fills it with DNA, after which the tail assembly is attached.

How does the DNA loading pump work? It is closely docked into the phage head structure, has a pentagonal structure attached to the phage head, and a loosely attached, 12-sided inner rosette that they describe as a sort of bearing or ball-race. The outer pentagon has an ATPase at each vertex, and these fire sequentially during the pumping mechanism. Each ATP advances the DNA by about two base pairs. Presumably the head has a structure that guides the DNA into regular loops around its inside walls. 

Structure of the dodecameric portion of the phage DNA pump, without the ATPase pentameric portion. Obviously, the DNA threads through the center.

In the diagram below (reference), three steps are shown. First, (a, top), the "I" ATPase node (red) is linked to the "J" and "A" rosette nodes. "A" is where the rosette hooks into the DNA (red). Next, the rosette is expanded a bit, bringing "A" out of register from "I" and "C" into register with "II". At the same time, "C" links to the DNA two base pairs down from where "A" latched into it. In the third step, the rosette squashes again, the DNA ends up raised by two base pairs, and the process can start all over. It is a bit of a sleeve/ratchet mechanism. They do not speculate at this point which of these steps is the power stroke- were the ATP is hydrolyzed. Getting only two base pairs into the head per ATP doesn't seem very efficient, but it is evidently at the end of packaging, when the pressure rises to extreme levels, where this pump shines. And it can get a 19,000 bp genome into a phage head in three minutes, (~100 bp per second), so it isn't a slouch when it comes to speed, either. 

Model of how this pump works. See text above for details.

Not only is this pump an amazing and powerful bit of biotechnology, able to compress DNA to sixty atmospheres, but it is a fourth fundamental type of motor, in addition to the rotary motors as found in flagella, the linear motors found along actin and microtubules, and the DNA threading/looping motors of condensin/cohesin.

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Who Needs the Fed?

Project 2025 promotes "free" banking, which is to say, no pesky regulations or backstopping from the Federal Reserve. What could go wrong?

Policy wonks can't help themselves- they need to write down their plans so that all the world can see how brilliant they are, and how real they could be, if only others recognized their brilliance. In that way, the project 2025 plans from the Heritage Foundation have been a gold mine, at least for Democrats. And since the Republican Party couldn't be bothered to write down a platform, other than "anything Trump wants", this project serves as the functional platform of the current Republican campaign, written as it was by scores of officials from the first Trump administration, plus many others itching to be appointed to a second. And it is crazy- more like a project 1825 than anything we would want to look forward to.

One of its less publicized planks is its approach to banking. It heaps criticism on the Federal Reserve, and recommends, as the most effective solution, its abolition and a return to "free banking". Which means a world where no regulator controls the banks, and no federal reserve backs it up against panics and crises. And just to complete the return to barbarism, it recommends a return to the gold standard as well.

"In free banking, neither interest rates nor the supply of money is controlled by the government. The Federal Reserve is effectively abolished, and the Department of the Treasury largely limits itself to handling the government’s money. Regions of the U.S. actually had a similar system, known as the “Suffolk System,” from 1824 until the 1850s, and it minimized both inflation and economic disruption while allowing lending to flourish." - From Chapter 4

Needless to say, US history is littered with banking panics, runs, and depressions, usually due to the unregulated nature of this "free" banking and to monetary gold backing. It is hard to express just how absurd and damaging it would be to return to such a world. The Federal Reserve was conjured up after a long history of the establishment of the first national bank, then its destruction by Andrew Jackson, a century of economic instability with particularly damaging panics in 1893 and 1907. By 1913, the US finally had had enough, and set up an updated national bank in the form of the Federal Reserve, to regulate and backstop the banking system. 

Illustration from 1873, portraying "Panic" on Wall Street.

Unfortunately, until the advent of Keynesian economics, it didn't really know what it was doing, and was particularly ineffective during the Great Depression, making things worse instead of better. Even now, it amounts to a cabal of bankers who are more interested in jacking up interest rates than in national prosperity. MMT economists tend to think that interest rates should be kept low, and the functions of the Federal Reserve folded into the Treasury Department, with greater political oversight. The use of interest rates- which are such a blunt tool of economic policy- could then be de-emphasized, in favor of more dynamic fiscal policy to manage inflation and monetary conditions. It is worth noting that over the last eighty years, the Fed has routinely over-shot its mark in raising interest rates, ending up with recessions, and rapid, belated retreats to lower rates. It is only with the current cycle that it has achieved, at least so far, the dream of a soft landing, taming inflation while avoiding recession.

Recessions (gray) have regularly followed interest raising campaigns by the Fed, and not always intentionally.

But note that the word "depression" is no longer in our lexicon. For all its faults, the Fed has kept the economy on a much more even keel than was possible under the wild-west free banking era, when monetary conditions were hostage to whatever Yosemite Sam dug up in the Yukon, or how wildly bankers over-extended their issuance of notes. Banks built impressive buildings to foster the illusion of stability in an environment where stability was impossible, lacking the infinite backstop that the Federal Reserve can now bring to bear during a crisis. Both individual depositors and the population as a whole benefit. It is a classic example of the people of the US coming together to create an institution that makes our lives better, so that we can worry about other things than the next banking panic. 

This economic craziness is just one small example of the fevered imaginations of the right wing in current US politics, which seems to have crawled out from the former fringes of Lyndon Larouche and the Birchers to take over an entire half of our political system. And this is fundamentally thanks to the air given them by an appalling right wing media that cares nothing for truth or civility, rather making its money from button pushing, whining grievance, and reflexive anti-state propaganda. And people complain about social media! Just how long ostensibly reasonable and decent (even Christian(!)) people will wallow in this environment is anyone's guess, but our common, rational, and beneficial institutions will in the mean time be in constant danger.

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Correction- The Republican convention did actually come up with a platform.