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ZoSaLa’s Innovative Approach

In general, molecules called proteins are the “worker and doer” molecules in cells and nucleic acids, usually called DNA (genes) or RNA carry the information needed for cells to make proteins. Different types of cells often have common and unique sets of active proteins operating at any given time. Indeed, the set of proteins, their quantity and their level of activity dictates in large part the type and function of a cell (cell phenotype), whether the cell is healthy and whether it is doing its job effectively. It is now accepted that the mere presence of DNA that encodes a particular protein is not definitive or even strong evidence that the corresponding protein is being made at all, or if made, is playing a significant role in a cell, at any given time.

 

Given the crucial role of proteins there has been a major effort to understand the molecular mechanisms involved in determining which proteins are active, their quantity and their level of activity. Over time a very important field of science has emerged called epigenetics which focusing on understanding precisely what mechanisms determine control over which genes are expressed, which proteins are made, which are active, and their quantity and their level of activity.

 

One exciting finding from epigenetic studies is that molecular mechanisms have evolved that enable cells to orchestrate changes in expression of panels of genes.  These mechanisms may be harnessed to promote healing, renewal and to improved function.

ZoSaLa’s new approach to treating aortic disease focuses on supporting and renewing populations of vascular smooth cells (vSMC) that are critical for aortic health.  The layers of the aortic wall are illustrated in the figure below. The maintenance of aortic wall integrity is managed primarily by the intrinsic vascular smooth muscle cells (vSMC) of the aortic wall, particularly vSMC in the middle layer (tunica media) of the wall. These cells demonstrate phenotypic and functional changes as part of aneurysmal disease progression. The transformation of vSCM phenotype that occurs during TAA and AAA, has been the subject of intense investigation and much is known that implicates vSCM pathology as the major culprit in AAA and many cases of TAA onset and progression. VSMCs in the healthy aortic wall display a contractile phenotype to maintain the vascular tone. In contrast, in the diseased aorta, vSCM are described as possessing a synthetic phenotype. Synthetic vSMC do not express the genes that encode the factors that impart aortic wall integrity and the ability to respond appropriately to changes in blood volume. ZoSaLa’s therapies reset genetic programing so that cells that form the aortic wall, especially endothelial cells which line the inner most layer in direct contact with blood flow and vSMC that reside in the middle layer, express genes associated with good health and function and turn off genes associated with pathology.

Figure 2: Layers of the aortic wall
Figure 3: Cartoon comparison healthy vs. diseased arotic wall

As depicted in figures 3 and 4 above, the current consensus view as to the ultimate cause of aortic aneurysmal disease is a breakdown and weakening of the layers of the aortic wall driven in large part by vSMC pathology. ZoSaLa’s academic collaborators, informed by decades of research into the causes of AAA and metabolic disease, were aware that diabetics are, surprisingly, protected against AAA. And they had previously shown that BHB protects aged vascular tissue against toxins that provoke cell death. These observations motivated them to test whether BHB might be protective in two commonly used animal models of AAA. Figures 5 and 6 are representative of the results of these studies.

Figure 5A-5C

They found that elevated BHB remarkably impedes aneurysmal expansion and, in some cases, fully reversed and healed the aneurysm. While some agents are, from time to time, reported to preclude induction of an aneurysm or slow its advance, reversal of established AAA is almost never seen in animal models. In the AAA animal studies conducted by ZoSaLa’s academic collaborators and the Zayed lab (Washington University), BHB exposure was elevated in rodent plasma by dissolving ketone ester D(R)-BHB in drinking water or feeding a ketogenic diet. Rats that received KD and exogenous BHB (KE) in the Zayed lab experiments, (see Sergio Sastriques-Dunlop, Santiago Elizondo Benedetto, Batool Arif, Rodrigo Meade, Mohamed S. Zaghloul, Sean J. English, Yongjian Liu,   Mohamed A. Zayed doi: https://doi.org/10.1101/2023.02.21.529460) reached a state of ketosis (BHB levels >0.5mM), and had significantly reduced AAA expansion and incidence of rupture. In the experiments done by ZoSaLa’s collaborators, BHB exposure was likewise well above that needed for ketosis (1.3mM-2mM). Most healthy persons on a normal, not a ketogenic diet, have a circulating plasma concentration of ~0.15mM BHB although the level varies across individuals and depends on diet and other factors.

Figure 6

The time course of AAA healing in the animal model studies, two- four weeks, appears sufficient to allow for renewal of the damaged aortic wall. And the data reflects that AAA induction results in senescent or synthetic vSMC, wall weakening and dilation. Over time, under BHB, therapy damaged cells are cleared out and healthy vSMC replace them. Investigation aimed at more precisely understanding how BHB exerts its profound healing impact is underway. It is well accepted that the “phenotype” of all cells, including vSMCs, is largely determined by the groups of genes that are active (expressed) or dormant in a cell at any given time. A healthy vSMC with a contractile phenotype is characterizes by the expression of a defined group of genes, while an unhealthy vSMC with a synthetic phenotype may be characterized by the expression of a different set or panel. In recent years, the area of science called epigenetics has begun to elucidate the intricate molecular genetic mechanisms that determine which genes are expressed, when. Simply stated, genetic material is tightly bundled as required for storage in cells. Processes that tend to loosen the wrapping, make certain genetic sequences more accessible for copying (expression). Retightening, once again, turns their expression down.  It has emerged that BHB is a salient factor in epigenetics. It especially plays a role in the molecular tools that relax gene binding to storage spool like proteins (“histones”) to enable expression. The level of BHB has both indirect and direct influences on which sets of genes are expressed inside cells. ZoSaLa’s approach is innovative and in line with cutting age research that unequivocally underscores that the failure of the vSMC in the middle layer of the aorta (tunica media) underlies TAA and AAA.  Nonetheless, it is critical to recognize that no animal model recapitulates human TAA or AAA. Some agents that have shown benefit in animal models in the past have not improved outcomes for TAA or AAA patients. Rigorous evaluation in TAA and AAA patients, is the only means to determine whether BHB will benefit patients.

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