Zhang, Bin

The structural protein, fibrillin, is important for maintaining the strength and reliability of connective tissue found in blood vessels and heart valves. Mutations in fibrillin‐1 result in Marfan syndrome and leads to left‐sided heart valve regurgitation, aortic root dilatation and aortic aneurysm. While previous studies have focused predominantly on young fibrillin‐mutant mice, hemodynamically significant disease generally does not emerge in patients with fibrillin mutations until later in life. Thus, this study aims to evaluate molecular and functional changes in heart valves from aged fibrillin mutant mice. Wild‐type (+/+) or fibrillin mutant (+/C1037G) mice were fed normal chow diet for 3 months (young) or 14 months (old). Heart valve function, aortic dimensions, and cardiac function were evaluated using high‐resolution echocardiography, and gene expression was measured using qRT‐PCR. Consistent with previous reports, we found no evidence of mitral regurgitation in young mice of either genotype. In old +/C1037G mice, however, we did find evidence of mitral regurgitation (~5%). In contrast, aortic valve regurgitation was evident in a relatively large proportion of +/C1037G mice at both ages (~20% at both time points), and was not detected in any +/+ animals. Interestingly, left ventricular dimensions were not associated with the presence of mitral regurgitation, but aortic root dilatation was present in all mice with aortic valve regurgitation. To gain insight into the molecular underpinnings of these phenotypic changes, we measured expression of IL‐10 (an anti‐inflammatory gene) and Runx2 (a pro‐proliferative gene that drives matrix remodeling), both of which are genes previously implicated in the pathogenesis of mitral and aortic valve disease. In young animals, expression of IL‐10 in mitral valves tended to be increased in +/C1037G mice compared to +/+ littermates. With aging, IL‐10 increased substantially in mitral valves from in +/+ mice, and increased further in +/C1037G littermates. Similar trends were observed in expression of Runx2 in mitral valves from both genotypes of mice. While IL‐10 followed a similar pattern in aortic valve tissue from young and old mice of both genotypes, Runx2 was not significantly increased across any of the groups. Collectively, while our data suggest that there are substantial, potentially adaptive molecular changes that occur during development of regurgitant valvular diseases, aortic dilatation may be a key early factor driving development of aortic regurgitation. Future experimental work aimed at determining the relative mechanical and molecular contributions to development of heart valve regurgitation will be critical to development of novel therapeutic strategies in patients with syndromes secondary to fibrillin mutations. Support or Funding Information NHLBI (R01‐HL111121)

Background While recent work from our laboratory showed that DNA (cytosine‐5)‐methyltransferase 3 beta (DNMT3b) is significantly increased in tissue from humans with calcific aortic valve stenosis and is associated with increases in global genomic methylation, it is unknown whether the net effects of these changes are protective or deleterious. The objective of this study was to test the hypothesis that genetic reduction of DNMT3b allows for increased anti‐osteogenic gene expression, and consequently results in suppression of osteogenic signaling and slower progression of valvular stenosis. Methods We used Ldlr −/− /ApoB 100/100 (LA) mice that were wild‐type for DNMT3b (LA‐DNMT3b +/+ ) or deficient in DNMT3b (LA‐DNMT3b +/− ) and fed a western diet for six months. Results Using quantitative real‐time RT‐PCR, we confirmed that expression of DNMT3b was reduced by ~50% in valves from haploinsufficient mice. In line with our hypothesis, reductions in DNMT3b were associated with increases in expression of FABP4 (which opposes osteogenesis) and Smad6 (which opposes canonical BMP signaling). Similarly, expression of the osteogenic genes Msx2 and Spp1 were substantially reduced in DNMT3b‐deficient mice. Critically, aortic valve cusp separation distance was significantly greater in DNMT3b‐deficient mice, demonstrating slower progression of valvular dysfunction. No other gross abnormalities were noted in the mice. Conclusions Collectively, these data suggest that selectively reducing DNMT3b activity may be a viable therapeutic approach to slowing progression of calcific aortic valve stenosis in humans. Support or Funding Information Grant Funding Source: Supported by NHLBI

Abstract Dynamic measures of resilience—the ability to resist and recover from a challenge—may be informative of the rate of aging before overt manifestations such as chronic disease, disability, and frailty. From this perspective mid-life resilience may predict longevity and late-life health. To test this hypothesis, we developed simple, reproducible, clinically relevant challenges, and outcome measures of physical resilience that revealed differences between and within age groups of genetically heterogeneous mice, and then examined associations between mid-life resilience and both lifespan and late-life measures of physiological function. We demonstrate that time to recovery from isoflurane anesthesia and weight change following a regimen of chemotherapy significantly differed among young, middle-aged, and older mice, and were more variable in older mice. Females that recovered faster than the median time from anesthesia (more resilient) at 12 months of age lived 8% longer than their counterparts, while more resilient males in mid-life exhibited better cardiac (fractional shortening and left ventricular volumes) and metabolic (glucose tolerance) function at 24 months of age. Moreover, female mice with less than the median weight loss at Day 3 of the cisplatin challenge lived 8% longer than those that lost more weight. In contrast, females who had more weight loss between Days 15 and 20 were relatively protected against early death. These data suggest that measures of physical resilience in mid-life may provide information about individual differences in aging, lifespan, and key parameters of late-life health.

Cardiovascular calcification is often an active process involving re-differentiation of cells to an osteoblast-like phenotype, and is strongly associated with aging. Sirtuins are histone and protein deacetylases that play a critical role in a number of age-related diseases, and SIRT6-deficient mice have the most profound progeroid phenotype. The role of SIRT6 in the regulation of cardiovascular calcification, however, is poorly understood. We hypothesized that age-related reductions in sirtuins promote osteogenic differentiation in cells from aortic valve and aorta. We used aortic valve interstitial cells isolated from SIRT6-deficient mice, pigs, or humans, and vascular smooth muscle cells from mouse aorta to determine whether osteogenic differentiation of cardiovascular cells is regulated by SIRT6 levels. In line with our hypothesis, we found increased p-SMAD1/5/8 levels in cells from SIRT6-deficient aortic valve and aorta. Second, global SIRT inhibition with NAD+-depletion increased histone acetylation and protein levels of the osteogenic proteins Sp7 and Runx2. Third, inhibition of SIRTs enhanced the transcriptional responses of Sp7 and Runx2 when cells were treated with BMP2. Fourth, knockdown of SIRT6 in mouse and human aorta and aortic valve cells by RNA interference increased Sp7 expression. Finally, chronic pharmacological inhibition of SIRTs promoted calcified nodule formation and calcium deposition in cultured mouse/swine aortic valve cells. Collectively, our data suggest that reductions in SIRT6 levels favor amplification of pro-osteogenic signaling and osteogenic differentiation of cardiovascular cells and promote the formation of calcified nodules in vitro . These data also suggest that sirtuin re-activation may be a useful therapy to slow initiation and/or progression of cardiovascular calcification in vivo.

Introduction While progression of calcific aortic valve stenosis (CAVS) is known to be strongly associated with activation of pro-inflammatory and pro-osteogenic pathways, effective treatments for CAVS have remained elusive. TNF-α is a pleiotropic inflammatory cytokine and is upregulated in patients with CAVS. Hence, we hypothesized that TNF-α inhibition, using the neutralizing antibody infliximab, can slow the progression of CAVS in vivo . Methods We used Ldlr -/- Apob 100/100 mice, which develop robust CAVS in an age-dependent manner, as a model. Mice were fed a Western-type diet for 6 months, then randomized into one of two groups: 1) twice-weekly infliximab injections (10 mg/kg intraperitoneally; n=10), and 2) saline injections (n=9). Aortic valve function was evaluated using echocardiography, and gene expression in aortic valves was measured using quantitative real-time PCR. Data are presented as mean ± SEM. Results During the 3 months preceding treatment initiation, aortic valve cusp separation distance decreased progressively in both groups (reduction by 0.082±0.011 mm in control group, 0.095±0.016 mm in infliximab group; p =0.27). Three months after initiation of treatment, cusp separation continued to decrease in control mice (by 0.108±0.021 mm), while progression of valve dysfunction was slower by 42% in the infliximab group (reduction of 0.063±0.009 mm; p =0.03 vs control). Expression of the pro-inflammatory genes Vcam1 and Nos2 was significantly reduced in valves of the infliximab group (by 37% and 60%, respectively). Moreover, endothelium-dependent vasodilation was improved by infliximab (improvement in maximum relaxation to acetylcholine in infliximab group 18±6% compared to control). Conclusions This study is the first to report that in vivo inhibition of TNF-α slows progression of CAVS in mice with established valve disease. This beneficial effect was accompanied by reduction in pro-inflammatory gene expression and improvement of systemic endothelial function, suggesting infliximab may exert its effects in part by increasing nitric oxide bioavailability. The findings warrant further investigation into the role of TNF-α in initiation and progression of CAVS, as well as its potential as a therapeutic target in human CAVS.

Previous studies have reported increases in oxidative stress due to reductions in antioxidant enzymes in calcified regions of stenotic human aortic valves. Whether reductions in antioxidant enzymes are adaptive, maladaptive, or an epiphenomenon in calcifying aortic valves remains unclear. Thus, the purpose of this study was to test whether overexpression of catalase reduces valvular calcification and slows progression of aortic valve stenosis in mice. Wild type (WT) mice with calcific aortic valve disease (ldlr-deficient, apoB100-only) were crossed with transgenic mice that overexpress human catalase (TG). All mice were fed a western diet (TD88137; Harlan Teklad). Functional echocardiogram data was collected and analyzed at early (3 months) and advanced (12 months) stages of valve disease, and valves were OCT embedded, sectioned, and stained for calcium levels (Alizarin Red). The transgenic valves show a slight increase in calcification compared to wild type counterparts. While there were no differences in valve function between genotypes at the early time point, cusp separation was decreased in the transgenic mice (0.74 ± 0.02 mm) compared to their wild-type littermates (0.84 ± 0.03 mm) at the 12 month time point (p < 0.05). In a subset of animals, valve calcification also tended to increase in transgenic mice at both time points compared to their wild-type littermates. In conclusion, overexpression of catalase resulted in a surprising finding of accelerated valvular calcification and stenosis. Interestingly, this finding aligns with the findings from several negative clinical trials testing antioxidant therapies in humans with atherosclerosis, suggests that the net impact of changing redox balance on disease progression are highly context dependent, and that increasing antioxidant capacity may not be a viable strategy to slow progression of calcific aortic valve disease in humans.

Activation of NADPH oxidase is a major contributor to hypertension, reactive oxygen species generation, endothelial dysfunction and increased vascular distensibility in angiotensin II (AngII)‐induced hypertension. Since exercise has been associated with increased antioxidant enzyme expression in blood vessels, we hypothesized that exercise training would improve endothelial function and vascular distensibility in mice with AngII‐induced hypertension. C57BL/6J mice received saline or AngII (1000 ng/kg/min) via osmotic minipumps, and were assigned to sedentary or treadmill exercise groups (13 m/min for 30 minutes, 5 days/week) for 14 days. Endothelium‐dependent relaxation in response to acetylcholine and arterial distensibility were assessed in carotid arteries using a pressurized arteriograph system. Gene expression was examined by qRT‐PCR. In AngII‐treated sedentary mice, endothelium‐dependent relaxation was impaired, while distensibility was higher than in saline‐infused sedentary mice. Compared to AngII‐infused sedentary mice, however, endothelium‐dependent relaxation was partially improved, and distensibility normalized in AngII‐infused exercised mice. In response to exercise, mRNA levels of SOD1 and SOD2 in aorta were increased, while mRNA levels of NOX2 were reduced. We conclude that exercise training can dramatically improve endothelial dysfunction and reverse derangements in carotid distensibility following prolonged AngII‐induced hypertension. Additional work examining mechanisms underlying our observed transcriptional changes will be critical to development of novel therapeutic targets.

The balance between local asymmetric dimethylarginine (ADMA) levels and dimethylarginine dimethylaminohydrolase 1 (DDAH1) activity is a major determinant of local nitric oxide synthase activity. Previous studies suggested that increased plasma ADMA levels are strongly associated with the aortic valve stenosis, causal or mechanistic links have yet to be elucidated. Thus, the aim of this study was to investigate whether reduction or overexpression of DDAH1 protects aortic valve dysfunction in mice. To experimentally alter DDAH1 levels, we used low density lipoprotein receptor deficient, apolipoprotein B100-only (LA) mice that were either DDAH1 wild-type (D +/+ or D 0/0 ), heterozygous (D +/- ), or transgenic/overexpressing (D Tg/0 ) littermates. All mice were fed a western diet for 6 months. Reducing DDAH1 increased plasma ADMA levels (LA-D +/+ =0.89 ± 0.02 μmol/L, LA-D +/- =1.1 ± 0.02 μmol/L, p< 0.05) and blood pressure (LA-D +/+ = 125 ± 4 mmHg, LA-D +/- = 141 ± 3 mmHg, p< 0.05) following 6 months of western diet feeding. Critically, aortic valve function was significantly worse in DDAH1-deficient mice (LA-D +/+ 2.20±0.09 m/sec versus LA-D +/- =2.41±0.11 m/sec, p< 0.05) and was associated with higher levels of valve cusp calcification (Alizarin Red, LA-D +/+ = 10 ± 3% versus LA-D +/- = 20 ± 4%). Reciprocally, overexpression of DDAH1 in hypercholesterolemic littermates reduced ADMA levels (LA-D 0/0 = 1.4 ± 0.2 μmol/L, LA-D Tg/0 = 0.7 ± 0.1μmol/L, p< 0.05) and systolic blood pressure (LA-D 0/0 = 136 ± 6 mmHg, LA-D Tg/0 = 124 ± 4 mmHg, p< 0.05). Interestingly, aortic valve function was not improved by overexpression of DDAH1 (LA-D 0/0 = 2.36 ± 0.06 m/sec versus LA-D Tg/0 mice (2.43 ± 0.21 m/sec), nor was valvular calcification. These results suggest that while endogenous levels of DDAH1 expression protect against increases in circulating ADMA, blood pressure, valvular calcification, and valvular dysfunction. Interestingly, overexpression of DDAH1 does not appear to confer additional protection against progression of valvular stenosis. Collectively, our data suggest effective therapeutic harnessing of the DDAH1/ADMA/NOS axis will require targeting of downstream effector molecules—such as oxidation state of soluble guanylate cyclase—known to be pathologically altered and rate limiting in a variety of cardiovascular diseases.

Heart transplantation is a surgical procedure performed on patients with end-stage heart failure or other irreversible heart disease, Heart transplant prolongs the life of severe heart disease patients. Most of the receipts could survive more than 2-3 years, five-year survival rate could reach 70-80% with immunosuppressive therapy, rejection still an important problem after transplantation. Currently, traditional calcineurin inhibitors, antimetabolite agents, and steroids, wildly used after transplantation, the new generation of immunosuppressive medicines have been developed, and cell-based immunotherapy, as mesenchymal stem cell, myeloid-derived suppressor cells, dendritic cells, pluripotent cells and Treg cells are promising to be used in cellular immunotherapy in organ transplantation.