May 10, 2012

Toward Regenerative Medicine Against Atherosclerosis cardiovascular disease

SENS Foundation-funded research shows that expression of a modified microbial enzyme protects human cells against 7-ketocholesterol toxicity, advancing research toward remediation of the foam cell and rejuvenation of the atherosclerotic artery.

Atherosclerotic cardiovascular disease is the principal cause of ischaemic heart disease, cerebrovascular disease, and peripheral vascular disease, making it the root of the leading cause of morbidity and mortality worldwide. Atherosclerosis begins with the entrapment and oxidation of low-density lipoprotein (LDL) cholesterol in the arterial endothelium. As a protective response, the endothelium recruits blood monocytes into the arterial wall, which differentiate and mature into active macrophages and engulf toxic oxidized cholesterol products (oxysterols) such as 7-ketocholesterol (7-KC). Although initially protective, this response ultimately leads to atherosclerotic plaque: oxidized cholesterol products accumulate in the macrophage lysosome, and impair the processing and trafficking of native cholesterol and other materials, leading macrophages to become dysfunctional and immobilized in the arterial intima. With ongoing entrapment of oxidized LDL in the tunica intima, more and more of these disabled "foam cells" progressively accumulate in the arterial wall, generating the fatty streaks that form the basis of the atherosclerotic lesion.

The results, although preliminary, are clearly promising for the potential of lysosomally-targeted DS1 cholesterol oxidase to remediate the oxysterol-intoxicated foam cell, and thus to potentially reverse the root cause of atherosclerosis. They also include some surprises that will need to be probed with further research. Why, for instance, was cell viability improved by DS1 ChOx/LAMP1 fusion protein even in cells that had not been treated with 7-KC (Fig. 1)? One possibility is that in addition to exogenous 7-KC, the enzyme degrades some basal level of oxysterol metabolites that are present even in untreated cells, or that it detoxifies other toxic lysosomal substrates. Another possibility, suggested in personal communication by J Mathieu, is that some metabolite of DS1 ChOx activity might actually provide a benefit to the cell. If such effects also emerge in vivo in any ultimate therapeutic use of suitably-modified DS1 ChOx, they might ultimately provide unexpected additional benefits to the functioning of the aging macrophage lysosome.

Rejuvenation biotechnology can be brought to bear against this disease of aging through the identification, modification, and therapeutic delivery of novel lysosomal enzymes derived from microbes to the arterial macrophage -- enzymes which are capable of degrading oxidized cholesterol products. SENS Foundation-funded researchers have been making steady progress in the identification and characterization of candidate enzymes for several years now, and a new report represents a substantial advance in the research: the rescue of cellular oxysterol toxicity by an introduced microbial lysosomal enzyme.

Previous research at the SENS Foundation Research Center and the LysoSENS project centered in Dr. Pedro Alvarez' lab at Rice University had identified several bacteria that express enzymes capable of degrading 7-KC, mostly in a manner consistent with 3β-oxidation to a ketone. Importantly, the product of such enzymatic transformations exhibited substantially reduced cytotoxicity to human embryonic kidney (HEK) cells relative to the parent molecule; unfortunately, however, there was little reason to expect that these enzymes would function in situ in the human foam cell lysosome, due to its substantially lower pH, high concentration of native proteases, and the derth of required cofactors.

Recently, however, Mathieu et al at Rice took note of a novel cholesterol oxidase from Chromobacterium sp. DS1, which exhibited "significant similarity (53-62%) to the cholesterol oxidases from Burkholderia spp. and Pseudomonas aeruginosa" and a "V(max)/K(m) ratio ... higher than those of commercially available cholesterol oxidases". Relative to other cholesterol oxidases, this enzyme exhibited very high stability in the face of heat, a range of organic solvents, and detergents, suggesting that it might well retain activity in the lysosome. Additionally, the enzyme's isomeric specificity prevented it from generating stereoisomer product species that permeabilize biological membranes.

Regenerative Medicine Against Atherosclerosis

Medicine has made remarkable progress against cardiovascular disease in recent decades, thanks to antihypertensive drugs and the advent of statins. But cardiovascular diseases remain the greatest cause of death and disease worldwide, and recent years have seen a series of high-profile failures to make further marginal gains over statins alone. Once-promizing agents such as ezetimibe, niacin, ApoA-1 Milano, and torcetrapib have either been found to provide no benefit, or evento increase mortality rates. These results suggest we may be reaching the limits of what can be achieved by pharmacological manipulation of metabolic pathways.

Although preliminary, these early results with the Chromobacterium sp. DS1 build on previous progress, carrying forward a clearly-defined developmental plan. If these results can be translated directly into the atherosclerotic artery, at minimum one would expect that the atherosclerotic process would be prevented or arrested. Infiltrating macrophages would for the first time be able to carry out their protective role in the artery, clearing out entrapped oxidized lipids without themselves ultimately falling to lysosomal toxicity and contributing to atherogenesis and the progression of advanced atherosclerotic lesions. Additionally, many ailing macrophages, at risk of becoming necrotic, would be have their functionality returned, their focal adhesions to the intima released, and would migrate out of the core of the lesion.

In an even more optimistic scenario, the abrogation of further insults to the arterial endothelium would be sufficient to allow the body's instrinsic regenerative capacity to finally catch up with and resolve other aspects of the arterial injury, clearing out the complex secondary pathology of even advanced atherosclerotic lesions and restoring the vessel to health. Aggressive use of statins and other radical lipid-lowering therapies have been demonstrated to shrink atherosclerotic lesions, but lipid-lowering alone has not proven sufficient to address many pathological features of advanced atherosclerotic lesions, including plaque calcification, fibrosis, endothelial erosion, extracellular lipid and lipoprotein deposits, cholesterol crystal formation and retention, and intraplaque neovascularization. Moving beyond risk-factor management and into the application of regenerative medicine to repair the damage in the aging artery offers the potential to finally heal the chronic injury of the aging artery.

With this research, SENS Foundation is making progress toward a new kind of therapy, applying the principles of regenerative medicine to the atherosclerotic lesion. This approach actually removes the molecular-level damage that underlies the disease, instead of attempting to perturb metabolism; to reverse the pathological basis of the disease, instead of merely bending the curb of its progression, or reducing the risk of individual cardiovascular events while leaving the disease itself unaddressed. In combination with other rejuvenation biotechnologies (such as the immunotherapeutic clearance of arterial lipoprotein amyloid and the restoration of arterial elasticity), this approach offers the potential to restore aging arteries to youthful health and functionality, allowing them to once again carry the blood of life safely and silently to rejuvenated hearts, muscles, kidneys, and brain.

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