Toward Regenerative Medicine Against Atherosclerosis

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.(1-3)

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.(3-8) SENS Foundation-funded researchers have been making steady progress in the identification and characterization of candidate enzymes for several years now,(6-8) 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;(6) 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”.(11) Relative to other cholesterol oxidases, this enzyme exhibited very high stability in the face of heat, a range of organic solvents, and detergents,(10) 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.

These properties commended the Chromobacterium sp. DS1 cholesterol oxidase (DS1 ChOx) to the Rice lysoSENS researchers as a potential candidate for degradation of toxic oxysterols trapped in the foam cell. Few cholesterol oxidases had been tested against 7-KC, and in their hands several bacterial enzymes had failed to oxidize it, or were only capable of doing so at unsuitably-high pH. But in preliminary testing, DS1 ChOx

proved active against both cholesterol and 7KC in detergent or mixed micelles. Overnight incubations of 7KC with DS1 ChOx resulted in complete elimination of 7KC as measured by HPLC (235.8 nm), with concomitant formation of a new product (319 nm). … Enzyme activity against both cholesterol and 7KC increased at higher substrate mole fractions. GC–MS of the HPLC fractions from the DS1 ChOx-catalyzed 7KC reaction revealed the presence of two large peaks, one at 472, which corresponds to the molecular weight of 7KC after derivatization, and the other with a molecular weight of 470 (Fig. 2C). This is supportive of 3β-oxidation, which is expected to result in the loss of two hydrogen atoms.(9)

To be used in vivo to remediate the oxysterol-congested lysosome, however, the enzyme would need to be targeted to, and retain activity in, the endosomal-lysosomal compartment of the cell. Accordingly, Mathieu et al generated a DS1 ChOx construct, utilizing the signal sequence and transmembrane domain of the lysosomal membrane glycoprotein lysosome-associated membrane protein-1 (LAMP-1). After confirming the lysomal localization of their construct when expressed in human fibroblasts,  Mathieu et al assessed the ability of the DS1 ChOx/LAMP1 fusion protein to alleviate the cytotoxicity of 7-KC in transfected, 7-KC-treated human fibroblasts. These effects were then compared to several competing approaches, each highlighting a specific property of the enzyme. These included  cytoplasmically-targeted DS1 ChOx; overexpression of LAMP-1 alone, without associated DS1 ChOx; sterol 27-hydroxylase, whose metabolites are known to be less toxic that 7-KC itself, but which is expressed in mitochondria rather than at the site of toxicity in the lysosome; and 11β-hydroxysteroid dehydrogenase, which is expressed in the endosome, but whose products are more cytotoxic than the 7-KC substrate itself.(9)