Citations

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.

Abstract Cellular senescence has been identified as a pathological mechanism linked to tau and amyloid beta (Aβ) accumulation in mouse models of Alzheimer's disease (AD). Clearance of senescent cells using the senolytic compounds dasatinib (D) and quercetin (Q) reduced neuropathological burden and improved clinically relevant outcomes in the mice. Herein, we conducted a vanguard open-label clinical trial of senolytic therapy for AD with the primary aim of evaluating central nervous system (CNS) penetrance, as well as exploratory data collection relevant to safety, feasibility, and efficacy. Participants with early-stage symptomatic AD were enrolled in an open-label, 12-week pilot study of intermittent orally-delivered D+Q. CNS penetrance was assessed by evaluating drug levels in cerebrospinal fluid (CSF) using high performance liquid chromatography with tandem mass spectrometry. Safety was continuously monitored with adverse event reporting, vitals, and laboratory work. Cognition, neuroimaging, and plasma and CSF biomarkers were assessed at baseline and post-treatment. Five participants (mean age: 76±5 years; 40% female) completed the trial. The treatment increased D and Q levels in the blood of all participants ranging from 12.7 to 73.5 ng/ml for D and 3.29-26.30 ng/ml for Q. D levels were detected in the CSF of four participants ranging from 0.281 to 0.536 ng/ml (t(4)=3.123, p=0.035); Q was not detected. Treatment was well-tolerated with no early discontinuation and six mild to moderate adverse events occurring across the study. Cognitive and neuroimaging endpoints did not significantly differ from baseline to post-treatment. CNS levels of IL-6 and GFAP increased from baseline to post-treatment (t(4)=3.913, p=008 and t(4)=3.354, p=0.028, respectively) concomitant with decreased levels of several cytokines and chemokines associated with senescence, and a trend toward higher levels of Aβ42 (t(4)=-2.338, p=0.079). Collectively the data indicate the CNS penetrance of D and provide preliminary support for the safety, tolerability, and feasibility of the intervention and suggest that astrocytes and Aβ may be particularly responsive to the treatment. While early results are promising, fully powered, placebo-controlled studies are needed to evaluate the potential of AD modification with the novel approach of targeting cellular senescence.

Increases in vascular calcification (VC) and fibrosis are a near ubiquitous finding with aging. Here, we sought to test the hypothesis that Metformin (MET)—an AMPK activator entering large clinical trials that is thought to attenuate multimorbidity associated with aging—attenuates key molecular processes known to drive vascular calcification (VC) and fibrogenic signaling in vascular smooth muscle cells (VSMCs) in vitro. Given recent reports highlighting the importance of mitochondria and sirtuins with aging, we also tested the hypothesis that MET-driven reductions in osteogenic signaling are associated with upregulation of SIRT3. We used exogenous BMP2 (100 ng/ml) and TGFβ1 (10 ng/ml) as osteogenic and fibrogenic stimuli, respectively. Consistent with prior work from our lab, treatment with BMP2 for 24 hours robustly increased expression of the osteogenic genes Msx2 (3.9±1.5 fold-change, FC), Runx2 (3.5±1.2 FC), and osterix (17.2±5.2 FC) in VSMCs. Critically, co-treatment with Metformin dramatically reduced expression of these genes by 68 ± 22%, 58 ±20%, and 63 ± 4%, respectively. Subsequent experiments treating cells with exogenous TGFβ1 resulted in significant induction of the fibrogenic marker/known TGFβ1 target versican (1.7±0.4 FC), an effect that was largely attenuated by co-treatment with MET (58±3%). Treatment with MET did not, however, impact TGFβ1-induced increases in CTGF or fibronectin. Treatment with either BMP2 or TGFβ1 resulted in significant increases in mRNA levels of SIRT3 (2.5 ±0.11 and 1.4 ±0.17 FC, respectively), whereas treatment with MET resulted in modest and less consistent increases in SIRT3 mRNA (1.3 ± 0.2 FC). Interestingly, however, co-treatment with BMP2+MET or TGFβ1+MET resulted in uniform suppression of BMP2/ TGFβ1-induced increases in SIRT3 (54±19% and 25±12%, respectively). Collectively, these data suggest that treatment with MET may be a viable strategy to attenuate age-associated increases in osteogenic and fibrogenic signaling in conduit vessels, and suggest further in vivo investigation into therapeutic efficacy is warranted.

Abstract Background and purpose: Poor ST-segment resolution (STR) is strongly associated with poor prognosis in patients with ST segment elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI). previous studies suggested higher HCY level in the people who live in high altitudes, so a retrospective analysis is conducted to examine the potential relationship between elevated serum HCY and poor STR after PPCI at high altitudes. Methods: This retrospective analysis included 308 high-altitude dwelling patients (1800-2200 meters elevation from the sea level) undergoing PPCI for STEMI during a period from September 2021 to March 2022. Clinical data were collected and statistically analyzed. Results: In comparison to the patients with normal plasma homocysteine (≤15 mmol/L; n = 155), patients with elevated homocysteine (>15 mmol/L) had higher percentage of men (92.81% vs. 80.00%; p = 0.001) and smoker (79.08% vs. 63.87%; p = 0.003), but no difference in other key baseline characteristics. The rate of complete ST-segment resolution after PPCI (≥ 70%) was 83.23% in the control group and 49.67% in the elevated HCY group (p ≤ 0.001). In multivariable regression analysis, poor ST-segment resolution (<70%) was independently associated with longer pain-to-balloon time (OR 0.832; 95%CI: 0.775–0.894), lower uric acid (OR 1.003; 95%CI: 1.000-1.005), and elevated HCY (OR 0.957 vs. normal HCY; 95%CI: 0.937–0.977). Conclusion: Elevated plasma HCY level was associated with poor ST segment resolution in patients undergoing PPCI STEMI at high altitude.

We recently reported activation of TGF-β superfamily pathways and activation of Wnt/β-catenin signaling in the pathogenesis of myxomatous mitral valve disease (MMVD), which may converge upon increased Runx2 expression. We also demonstrated that chronic in vivo administration of angiotensin II (AngII) recaptures various molecular aberrations characteristic of human disease -including increased TGF-β signaling and activation of Runx2- in mouse mitral valves. Here, we sought to determine whether Runx2 haploinsufficiency can attenuate molecular and/or phenotypic effects of AngII infusion in mouse mitral valves. We implanted littermate matched Runx2+/+ and Runx2+/- mice with osmotic minipumps delivering AngII (1000ng/kg/min) or saline for 2 weeks. We measured changes in mRNA (qRT-PCR) and protein (immunohistochemistry) levels in mitral valves, and assessed mitral valve function by echocardiography. In Runx2+/+ mice, AngII elicited increased mRNA levels of TGF-β3 and the TGF-β target COL3A1, and increased staining for canonical (p-SMAD2/3) and non-canonical (p-AKT) TGF-β signaling proteins. Runx2 haploinsufficiency attenuated AngII-induced changes in COL3A1 expression and p-AKT staining, without impacting AngII-mediated alterations in TGF-β3 and p-SMAD2/3. Evaluating collagen thickness by picrosirius red staining and visualization under circularly polarized light, AngII infusion in Runx2+/+ mice was associated with increased collagen density at the base of posterior mitral valve leaflets, a change that was abrogated by Runx2 haploinsufficiency. Similarly, Runx2-haploinsufficiency was again protective when examining mRNA and protein levels of the proliferation marker KI67 in response to AngII. Echocardiography identified a trend towards increased mitral valve regurgitation following AngII infusion that was unaltered by Runx2 haploinsuffiency. Collectively, these data suggest that increases in Runx2 expression in the mitral valve may serve to orchestrate tissue fibrosis and cellular proliferation that are hallmarks of MMVD, and that future pharmacological strategies aimed at reducing activity of Runx2 in the mitral valve may yield viable non-surgical alternatives for the management of this condition.

Mitochondrial antioxidant genes are major regulators of oxidative stress in the vasculature. Previous data has shown that reducing antioxidant capacity accelerates atherosclerotic plaque size, however, it is unclear whether reducing mitochondrial antioxidant capacity alters plaque composition. Therefore, we tested the hypothesis that reductions of mitochondrial antioxidant capacity will increase osteogenic markers and intimal plaque calcification in aorta from hypercholesterolemic mice. We used Ldlr -/- ApoB 100/100 (LA) mice that were either wild-type (LA-MnSOD +/+ ) or heterozygous (LA-MnSOD +/- ) for manganese superoxide dismutase that were fed a Western Diet (TD88137) for 3 or 6 months. Changes in changes gene expression were assessed in aortic arch using qRT-PCR, plaque calcium levels were examined using histological evaluation from aortic cryosections, and endothelial function was measured using isolated organ bath chambers ( ex vivo ). While expression of SP7 (an osteogenic transcription factor) was unchanged between LA-MnSOD +/+ (1.0±0.6) and LA-MnSOD +/- (0.95±0.3) at 3 months, SP7 levels were dramatically increased in LA-MnSOD +/+ (8.0±2.8) at 6 months and increased further in LA-MnSOD +/+ mice (17.5±5.9; p<0.05). Alizarin Red staining for calcium showed increased calcium burden in plaques of 6 month LA-MnSOD +/- compared to LA-MnSOD +/+ (3636±669 pixels versus 649±206 pixels; p<0.05). Vasomotor function in aorta was unchanged between LA-MnSOD +/+ and LA-MnSOD +/- mice, suggesting that the transcriptional and functional changes related to calcification in LA-MnSOD +/- mice occur independently of nitric oxide signaling. In conclusion, our data strongly support our working hypothesis that reductions in mitochondrial antioxidant capacity significantly accelerate osteogenic calcification in aorta from hypercholesterolemic mice, and suggest that targeting mitochondrial oxidative stress may be a viable therapeutic strategy to slow vascular calcification.

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.

Thrombin plays a major role in coagulation cascade and participates in regulation of vascular function through its effects on protease-activated receptors (PARs). The effect of thrombin inhibition on vascular and aortic valve function during prolonged hypercholesterolemia, however, remains unknown. In the present study, we tested the hypothesis that direct thrombin inhibition (DTI, dabigatran etexilate) slows progression of endothelial and aortic valve dysfunction in ldlr-deficient, apolipoprotein B100-only (LA) mice. Carotid arteries from LA mice were used for studies of endothelial function in isolated organ baths, and aortic valve function was evaluated using echocardiography. In placebo-treated mice, maximum relaxation to acetylcholine was reduced following 6 and 9 months of hypercholesterolemia (maximum relaxation to acetylcholine (MR ACH ) =75%± 3.7, and 67%± 3.6, respectively), but not affected by DTI (75%± 2.5, and 62%± 3.2, respectively). Relaxation to exogenous NO donors was also unaffected by DTI. Aortic valve function progressively deteriorated at 3, 6, and 9 months in placebo-treated mice (2.1 m/s± 0.04, 2.5 m/s± 0.1, and 2.9m/s ± 0.1, respectively), and was not affected by DTI (2.0m/s± 0.1, 2.5m/s± 0.1 and 3.0m/s ± 0.4, respectively). While PAR receptor expression was detected in aorta and aortic valve tissue, expression of SPHK1 (an indicator of PAR1 activity) was not altered by DTI. Expression of CTGF in aortic valves, however, tended to be reduced by DTI. In conclusion, our data suggest that DTI does not prevent vasomotor or aortic valve dysfunction in mice with prolonged hypercholesterolemia, but may attenuate pro-fibrotic signaling in aortic valves.

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.

Increases in mitochondrial reactive oxygen species (ROS) can contribute to endothelial dysfunction in conduit arteries by reducing nitric oxide bioavailability. SIRT3 is a protein deacetylase localized to mitochondria and regulates activity of numerous electron transport chain enzymes, and can be inactivated by increases in mitochondrial ROS. Whether reductions in SIRT3 are an independent contributor to endothelial dysfunction, however, are unknown. Therefore, we hypothesized that SIRT3 null mice (SIRT3 -/- ) will have impaired relaxation to acetylcholine (MR Ach ) compared to wild-type littermates (SIRT3 +/+ ), and that this difference will be exacerbated by Western diet feeding for 3 months (TD88137, 42% of kcal from fat, 0.2% cholesterol). Endothelial function was measured using isolated organ bath chambers, and endothelium-dependent (MR Ach ) and endothelium-independent (sodium nitroprusside, MR SNP ) relaxation were measured. In contrast to our hypothesis, MR ACH was virtually identical between SIRT3 +/+ and SIRT3 -/- fed a standard chow diet (MR ACH = 68±2% and 69±3%; respectively), and MR SNP also remained unchanged between the two genotypes. In groups fed a Western diet, however, endothelium-dependent relaxation was significantly impaired in SIRT3 -/- mice compared to SIRT3 +/+ littermates (MR ACH = 49±4% and 58±3%, respectively; p<0.05). MR SNP was not altered by either Western diet feeding or SIRT3 deficiency. Interestingly, impairments in MR Ach in SIRT3 -/- mice fed a Western diet were not rescued with acute incubation of apocynin (an NAD(P)H oxidase inhibitor) or mito-tempol (a mitochondria-targeted antioxidant), suggesting that chronic antioxidant and/or acetyltransferase inhibition may be required to improve vasomotor function in this model. Collectively, our data suggest that SIRT3 plays a major role in regulating endothelial function with dietary stress, and may be a useful therapeutic target to reduce cardiovascular risk.