Zhang, Bin

While the strong connection between oxidative stress and atherosclerosis has many to hypothesize that antioxidant therapy may be viable therapeutic target in combating progression of atherosclerosis, numerous clinical trials of antioxidant interventions that scavenge multiple reactive oxygen species have been negative. Here, we test the hypothesis that overexpression of catalase—an enzyme which selectively degrades H 2 O 2 —plays a protective role against the initiation and expansion of calcific lesions in advanced atherosclerotic plaques. In vitro, we used aortic smooth muscle cells (SMC) from Ldlr -/- ApoB 100/100 (LA) mice that were either wild-type for human catalase (LA-Cat 0/0 ) or overexpressing human catalase (LA-Cat Tg/0 ). After treatment with BMP2 or TNFα for 24 hours, we found that catalase-overexpressing VSMCs had modest reductions in TNFα-induced inflammatory markers but paradoxical increases in mRNA levels of BMP2-induced osterix and the SMAD-specific target ID1. To determine if these effects were conserved in vivo, we analyzed aortic tissue from LA mice fed a Western diet for 3 or 12 months. Surprisingly—and unlike our cell-based experiments—aorta from LA-Cat Tg/0 tended to have higher expression of inflammatory markers compared to their LA-Cat 0/0 littermates. Similarly, expression of osterix was increased in LA-Cat Tg/0 mice compare to their LA-Cat 0/0 littermates, which was also associated with an increase in intimal plaque calcification. Collectively, these data strongly suggest that systemically overexpression of catalase (a close parallel to systemic administration of a selective H 2 O 2 -scavenging antioxidant) does not result in molecular, histopathological, or functional benefits in large arteries from hypercholesterolemic mice. Future studies aimed at characterizing the complex interplay between reactive oxygen species and specific intracellular signaling pathways are an essential next step to understanding the role of endogenous reactive oxygen species in the pathogenesis of atherosclerotic vascular disease.

Objective: We recently discovered that activation of oxidized soluble guanylate cyclase with ataciguat (ATA) slows valve calcification and dysfunction in mice and humans with moderate aortic valve stenosis (AVS). We examined if ATA would attenuate multiple mechanisms contributing to AVS and end organ dysfunction associated with AVS, and hypothesized: 1) ATA attenuates osteogenic signaling in valve tissue from mice with AVS, thereby attenuating calcification and valve dysfunction, 2) AVS increases rates of sudden cardiac death in our model, and 3) treatment with ATA prevents QT prolongation and deleterious electrical remodeling commonly observed in AVS. Methods: Functional, molecular, and histopathological changes in valve tissue along with QT interval duration were measured in 3 independent groups of mice: non-AVS, AVS, and AVS+ATA (WD for 6 months then WD+ATA for 3 months (AVS+ATA). We gave 5 mg/kg isoproterenol (ISO, i.p.) to ldlr -/- /ApoB100-only mice fed normal chow (non-AVS) or western diet (AVS, fed WD for 9 months) to assess risk for malignant arrhythmias. Retrospective analysis of QT interval was also done on data from our recent Phase 1 clinical trial. Results: AVS+ATA mice had less valve calcification and slower progression of valve dysfunction through attenuation of canonical BMP signaling and reductions in Runx2 and SPP1 compared to AVS mice. QT prolongation was evident in AVS mice, and was significantly attenuated by treatment with ATA (p= 0.001) and coarsely associated with severity of valvular or ventricular function. While all non-AVS mice survived for 24 hours after ISO, 40% of mice with AVS reached a moribund equivalent within 4 hours. Retrospective analyses also showed reductions in QT interval duration in patients treated with 200 mg/day ATA for 14 days (p < 0.05). Conclusion: Collectively, this is the first study reporting increased propensity for malignant arrhythmias in our mouse model of AVS, QT prolongation as a likely underlying mechanism, and restoration of cardiac conduction dynamics with targeted restoration of nitric oxide signaling. Future studies focused on the role of sGC in the evolution and prevention of AVS and consequent deleterious electrical remodeling/malignant arrhythmia risk is warranted.

Background: Plaque fibrosis and re-differentiation of vascular smooth muscle cells (VSMCs) from a contractile to a “synthetic” phenotype are hallmarks of advanced atherosclerosis. While in vitro studies demonstrated a clear role of oxidative stress in the loss of VSMC contractile phenotypes, the roles of specific antioxidant mechanisms and their therapeutic utility remain unclear. Here we test the hypothesis that overexpression of catalase protects against excess plaque fibrosis and losses in contractile phenotypes in vivo . Methods: We used littermate-matched, hypercholesteremic (ldlr -/- /ApoB 100/100 ; LA) mice lacking a transgenic construct (LA-CAT 0/0 ) or harboring a single copy of the human catalase gene (LA-CAT Tg/0 ), which were fed a Western Diet for 3 or 12 months. Isolated organ bath chambers, histopathological analyses, and quantitative RT-PCR were used to assess vasomotor function, structure, and molecular changes in aorta. Results: Oxidative stress increased with disease progression in aortic tissue from LA-CAT 0/0 mice and was modestly reduced in LA-CAT Tg/0 mice. Collagen deposition and fiber thickness increased with disease progression in LA-CAT 0/0 mice, and these increases in fiber thickness were attenuated in LA-CAT Tg/0 mice at 12 months and more pronounced in male mice. In parallel, we observed favorable rightward shifts in the vascular stress-strain relationship in LA-CAT Tg/0 mice that were more pronounced in aged male mice. Interestingly, these structural changes were associated with improved contractile responses to either PGF 2α or serotonin in male LA-CAT Tg/0 mice at 12 months. Consistent with these changes, the contractile marker calponin-1 was profoundly reduced with increasing age in LA-CAT 0/0 mice, with these changes being partially attenuated in aging, male LA-CAT Tg/0 mice. In vitro, treatment of VSMCs with PEG-catalase attenuated BMP2-dependent contractile protein expression. Conclusion: These results demonstrate that moderate overexpression of catalase can attenuate deleterious fibrotic and contractile changes associated with prolonged progression of atherosclerosis. Future mechanistic investigation exploring the context-dependence of these changes (e.g., sex dependence) is warranted.

Introduction: Senescent cell accumulation and senescence-associated secretory phenotype (SASP) are putative major mechanisms of tissue aging, and growing evidence suggests that pharmacological clearance of senescent cells slows progression of multiple age-related diseases. Here, we hypothesized that treating with a known senolytic, Fisetin (FIS), would reduce molecular drivers of fibrosis and calcification and ultimately slow disease progression of fibrocalcific aortic valve stenosis (FCAVS) in mice when initiated in mild/moderate stages of disease. Methods: Male and female Ldlr -/- / ApoB 100/100 mice were fed a western diet for 9 months and assigned to one of two treatment groups: FIS or vehicle, with treatment beginning at 6 months of diet. Results: Treatment with FIS reduced CDKN2A (p < 0.01) and SASP marker TNFα (p < 0.002) in both sexes, and consistent with our hypothesis was associated with reduced expression of the osteogenic markers RUNX2 (p < 0.021) and SPP1 (p < 0.006) in valve tissue from female mice. In contrast, expression of WNT5A was reduced in valves only from male mice (p < 0.004). In line with these molecular changes, valvular calcium burden (alizarin red staining) also tended to decrease in both sexes. Interestingly, TGFβ1 expression was significantly reduced (p < 0.018) in both sexes and was associated with reductions in collagen 3A1. These molecular changes in fibrogenic signaling manifested as a shift towards thinner collagen fibers only in male animals (p < 0.05). Functionally, slower progression of valvular dysfunction (declines in aortic valve cusp separation distance assessed with high-resolution echocardiography) was observed predominantly in females (p < 0.002). Conclusions: In conclusion, these data suggest that long-term, intermittent clearance of senescent cells robustly attenuates osteogenic and fibrogenic signaling in mild/moderate FCAVS, although the molecular, histopathological, and functional consequences of senolytic therapy may occur in a sex-specific manner. Future studies evaluating preclinical efficacy of senolytics should account for tissue and sex specificity and warrant longer-term treatment to assess the functional consequences of the biological and histopathological changes observed here.

Increased oxidative stress is associated with initiation and progression of atherosclerosis. While catalase catalyzes the breakdown of hydrogen peroxide (H 2 O 2 ) to water, it is unclear whether overexpression of catalase is sufficient to protect against cardiovascular dysfunction and advanced plaque formation. Therefore, we hypothesized overexpression of catalase in hypercholesterolemic mice following 3 months or 12 months of Western diet feeding will have preserved endothelial function, reduced H 2 O 2 levels, reduced levels of osteogenic markers, and reduced calcium burden in atherosclerotic plaques. We used Ldlr − / − ApoB 100/100 (LA) mice (hypercholesterolemic mice with a more “humanized” lipid profile) that were either expressed endogenous levels of murine catalase (LA‐Cat 0/0 ) or overexpressed one copy of a human catalase construct (LA‐Cat Tg/0 ). These mice were fed a Western Diet (TD88137) for either 3 months (early disease) or 12 months (late disease). Following 3 months or 12 months on diet, we assessed endothelial function using isolated organ chambers, quantified H 2 O 2 production (staining of frozen cross‐sections of aorta with CM‐H2DCFDA), measured changes of gene expression in aortic arch (qRT‐PCR), and performed histopathological analyses of intimal plaque calcium burden (Alizarin Red). Following 3 months on diet, endothelium‐dependent relaxation to acetylcholine (ACH) was unchanged between LA‐Cat 0/0 and LA‐Cat Tg/0 mice, and we did not find substantive molecular or histopathological differences in aortic tissue between the two genotypes. Interestingly, and contrast to our hypothesis, endothelium‐dependent relaxation was modestly but not significantly impaired in LA‐Cat Tg/0 mice compared to LA‐Cat 0/0 mice after 12 months of western diet feeding. Evaluation of female mice alone, however, revealed profoundly impaired endothelium‐dependent relaxation in LA‐Cat Tg/0 . While H 2 O 2 levels were slightly reduced in LA‐Cat Tg/0 at 3 months, H 2 O 2 levels were not reduced by catalase overexpression following 12 months of Western diet feeding. In further contrast to our original hypothesis, expression of the osteogenic genes osterix and osteopontin were modestly increased in LA‐Cat Tg/0 mice following 3 months on diet compared to their wild‐type littermates, and further increased at the 12 month time point. Functionally, the molecular changes in 12 month animals were associated with increases in intimal plaque calcium burden in LA‐Cat Tg/0 (12.3% ± 2.5%) compared to their wild‐type littermates (7.8% ±1.2%). In conclusion, these data are in line with previous negative clinical trials of antioxidant treatment strategies, and suggest targeting a single antioxidant enzyme/reactive oxygen species to delay initiation or slow progression of atherosclerosis is not a viable therapeutic target, and may in fact paradoxically lead to deleterious changes in plaque composition and endothelial dysfunction. Support or Funding Information NHLBI HL111121 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .