Age-related Aortic Stiffness Can Be Transferred and Ameliorated via Fecal Microbiota Transplant in Mice

Age-related increases in aortic stiffness contribute to the development of cardiovascular diseases (CVD). To determine whether the gut microbiome (GM) modulates age-related aortic stiffening, we performed fecal microbiota transplants (FMT) between young (Y; 3 month) and older (O; 25 month) male C57BL/6N mice. Following antibiotic treatment (to suppress endogenous microbiota), mice received weekly FMT (fecal samples collected at baseline) via oral gavage for 8-16 weeks from their own (i.e., sham condition: Y-y, O-o [RECIPIENT-donor]) or opposite age group (Y-o, O-y) (N=8-12/group). In vivo aortic stiffness (pulse wave velocity [PWV]) was higher in older vs. young mice at baseline (382±8 vs. 328±7cm/sec, mean±SE, P<0.001). Arterial phenotypes were transferred such that old microbiota transplanted into young mice increased, while young into old decreased, PWV (Y-y: 325±10 vs. Y-o: 362±10cm/sec, P=0.022; O-o: 409±10 vs. O-y: 335±6cm/sec, P<0.001). Intrinsic mechanical stiffness of excised aortic rings (elastic modulus) increased after transplant of old into young (Y-y: 2141±223 vs. Y-o: 3218±394kPA, P=0.022), and decreased with young into old (O-O: 3263±217 vs. O-y: 2602±136kPA, P=0.016), indicating the GM mediates aortic stiffening by modulating structural changes in the arterial wall. Age-related increases in aortic abundance of advanced glycation end products (AGEs), which cross-link arterial structural proteins, tended to be transferred by the GM (Y-y: 0.022±0.001 vs. Y-o: 0.038±0.006 A.U., P=0.11; O-o: 0.120±0.029 vs. O-y: 0.038±0.009 A.U., P=0.06). The aging GM can induce aortic stiffening via promoting AGEs accumulation and crosslinking of arterial structural proteins, and thus might be a promising target for preventing/treating age-related aortic stiffening and CVD.

Is It Good to Have a Stiff Aorta with Aging? Causes and Consequences

Aortic stiffness increases with advancing age more than doubling during the human lifespan and is a robust predictor of cardiovascular disease (CVD) clinical events independent of traditional risk factors. The aorta increases in diameter and length to accommodate growing body size and cardiac output in youth, but in middle- and older age the aorta continues to remodel to a larger diameter thinning the pool of permanent elastin fibers increasing intramural wall stress resulting in the transfer of load bearing onto stiffer collagen fibers. While aortic stiffening in early middle-age may be a compensatory mechanism to normalize intramural wall stress and therefore theoretically 'good' early in the lifespan, the negative clinical consequences of accelerated aortic stiffening beyond middle-age far outweigh any earlier physiological benefit. Indeed, aortic stiffness and the loss of the "Windkessel effect" with advancing age results in elevated pulsatile pressure and flow in downstream microvasculature that is associated with subclinical damage to high flow, low resistance organs such as brain, kidney, retina and heart. The mechanisms of aortic stiffness include alterations in extracellular matrix proteins (collagen deposition, elastin fragmentation), increased vasodilator tone (oxidative stress and inflammation-related reduced vasodilators and augmented vasoconstrictors; enhanced sympathetic activity), arterial calcification, vascular smooth muscle cell stiffness and extracellular matrix glycosaminoglycans. Given the rapidly aging population of the U.S., aortic stiffening will likely contribute to substantial CVD burden over the next 2-3 decades unless new therapeutic targets and interventions are identified to prevent the potential avalanche of clinical sequelae related to age-related aortic stiffness.

Vascular Stiffening Mediated by Rho‐Associated Coiled‐Coil Containing Kinase Isoforms

Background The pathogenesis of vascular stiffening and hypertension is marked by non‐compliance of vessel wall because of deposition of collagen fibers, loss of elastin fibers, and increased vascular thickening. Rho/Rho‐associated coiled‐coil containing kinases 1 and 2 (ROCK1 and ROCK2) have been shown to regulate cellular contraction and vascular remodeling. However, the role of ROCK isoforms in mediating pathogenesis of vascular stiffening and hypertension is not known. Methods and Results Hemizygous Rock mice ( Rock1 +/− and Rock2 +/− ) were used to determine the role of ROCK1 and ROCK2 in age‐related vascular dysfunction. Both ROCK activity and aortic stiffness increased to a greater extent with age in wild‐type mice compared with that of Rock1 +/− and Rock2 +/− mice. As a model for age‐related vascular stiffening, we administered angiotensin II (500 ng/kg per minute) combined with nitric oxide synthase inhibitor, L‐N ω ‐nitroarginine methyl ester (0.5 g/L) for 4 weeks to 12‐week‐old male Rock1 +/− and Rock2 +/− mice. Similar to advancing age, angiotensin II/L‐N ω ‐nitroarginine methyl ester caused increased blood pressure, aortic stiffening, and vascular remodeling, which were attenuated in Rock2 +/− , and to a lesser extent, Rock1 +/− mice. The reduction of aortic stiffening in Rock2 +/− mice was accompanied by decreased collagen deposition, relatively preserved elastin content, and less aortic wall hypertrophy. Indeed, the upregulation of collagen I by transforming growth factor‐β1 or angiotensin II was greatly attenuated in Rock2 −/− mouse embryonic fibroblasts. Conclusions These findings indicate that ROCK1 and ROCK2 mediate both age‐related and pharmacologically induced aortic stiffening, and suggest that inhibition of ROCK2, and to a lesser extent ROCK1, may have therapeutic benefits in preventing age‐related vascular stiffening.

Abstract 13: P2X7 Receptor Knockout Attenuates Angiotensin II-Induced Hypertension, Vascular Injury And CD8 + T Cell Infiltration

Background: The P2X7 receptor (P2RX7) recognizes damage associated molecule patterns such as adenosine triphosphate (ATP), and triggers the activation of immune cells. Elevated plasma ATP levels have been observed in hypertensive patients, providing a potential mechanism for P2RX7 activation. Additionally, a hypomorphic polymorphism for P2X7 is correlated with a decreased risk for essential hypertension in Chinese post-menopausal women. However, it is unknown whether P2RX7 activation contributes to angiotensin (Ang) II-induced blood pressure (BP) elevation and vascular damage. We hypothesized that P2rx7 knockout would blunt Ang II-induced BP elevation, vascular injury, and infiltration of activated immune T cells into perivascular adipose tissue (PVAT). Methods: Ten-to-12-week-old male C57BL/6J male wild-type (WT) and P2rx7 -/- mice were infused or not with Ang II (1000ng/kg/min) for 14 days. BP was determined by telemetry, mesenteric artery function and remodeling using pressurized myography, aortic stiffening by ultrasound and infiltration of activated immune T cells in aortic PVAT by flow cytometry. Results: Ang II-infused P2rx7 -/- mice display a reduced systolic BP (164±3 vs. 176±2 mm Hg, P <0.05) and pulse pressure (37±4 vs. 53±3 mm Hg, P <0.001) in comparison to WT mice. Aortic stiffening occurred in WT mice treated with Ang II, demonstrated by an increased pulse wave velocity (7.7±0.7 vs. 5.9±0.3 m/s, P <0.05), accompanied by a 3.8-fold increased infiltration of activated CD8 + T cells in aortic PVAT (60±16 vs 16±3 cells/aortic PVAT, P <0.001), which were both absent in P2rx7 -/- mice (6.4±1.4 vs 5.5±1.1 m/s and 27±7 vs 16±3 cells/aortic PVAT). In addition, the frequency of IFN-γ producing CD8 + T cells in the spleen of Ang II-treated WT mice increased (2.6±0.2% vs 1.2±0.2%), which did not occur in P2rx7 -/- mice (1.7±0.3% vs 1.7±0.2%). Ang II-infusion induced mesenteric artery endothelial dysfunction in WT mice (61±7 vs 83±4% relaxation response to acetylcholine, P <0.05), which was absent in P2rx7 -/- mice (89±3 vs 90±3%). Conclusion: P2rx7 knockout attenuates Ang II-induced hypertension, vascular injury, and infiltration of activated CD8 + T cells into aortic PVAT.

Abstract P274: Inhibition Of Abnormal Exosome Release Prevents Excessive Aortic Stiffness And Relaxation Dysfunction In Diet-induced Obesity

Interactions between over-nutrition and abnormal exosome release impact insulin sensitivity and the development of cardiovascular disease (CVD). Recent data have shown that exosomes can be released from various cell types, including adipocytes and vascular cells, and that they exist in body fluids and tissues functioning as mediators of cell-cell communication. However, the specific role of exosomes in diet-induced excessive vascular stiffness and hypertension has not been explored. Accordingly, we hypothesized that abnormal release of exosomes contributes to western diet (WD)- induced aortic stiffening and impaired vascular diastolic relaxation. We further posited that GW4869, an antagonist of neutral sphingomyelinase 2 (nSMase2) which promotes exosome production and release, would prevent WD-induced aortic stiffening and impaired vascular relaxation. Six week-old female C57BL/6L mice were fed a mouse chow (CD) or WD containing excess fat (46%) and fructose (17.5%) for 16 weeks with or without GW4849. To this point, 200 μl of 0.3 mg/mL GW4869 in 0.9% normal saline (60 μg/mouse; 2-2.5 μg/g body weight) was injected intraperitoneally every 48 hours for 12 weeks. 16 weeks of WD induced an increase of aortic stiffness as examined by pulse wave velocity (PWV) and impaired the aortic vasodilation responses to acetylcholine (Ach) and sodium nitroprusside (SNP) (10 -9 -10 -4 mol/L). However, GW4869 treatment prevented the WD-induced excessive aortic stiffness, as well as impairment of endothelium dependent/independent vascular relaxation. There were no significant differences in blood pressure between each group examined by tail cuff blood pressure measurement. These findings support the hypothesis that abnormal release of exosomes play an important role in WD-induced excessive aortic stiffness, impaired vascular relaxation and CVD in diet-induced obesity.

Apigenin restores endothelial function by ameliorating oxidative stress, reverses aortic stiffening, and mitigates vascular inflammation with aging

We assessed the efficacy of oral supplementation with the flavanoid apigenin on arterial function during aging and identified critical mechanisms of action. Young (6 months) and old (27 months) C57BL/6N mice (model of arterial aging) consumed drinking water containing vehicle (0.2% carboxymethylcellulose; 10 young, 7 old) or apigenin (0.5 mg/ml in vehicle; 10 young, 9 old) for 6 weeks. In vehicle-treated animals, isolated carotid artery endothelium-dependent dilation (EDD), bioassay of endothelial function, was impaired in old vs young (70±9 vs 92±1 %, P<0.0001) due to reduced nitric oxide (NO) bioavailability. Old mice had greater arterial reactive oxygen species (ROS) production and oxidative stress (higher nitrotyrosine) associated with greater nicotinamide adenine dinucleotide phosphate oxidase (oxidant enzyme) and lower superoxide dismutase 1 and 2 (antioxidant enzymes); ex vivo administration of TEMPOL (antioxidant) restored EDD to young levels, indicating ROS-mediated suppression of EDD. Old animals also had greater aortic stiffness as indicated by higher aortic pulse wave velocity (PWV, 434±9 vs 346±5 cm/sec, P<0.0001) due to greater intrinsic aortic wall stiffness associated with lower elastin levels and higher collagen, advanced glycation end-products (AGEs) and pro-inflammatory cytokine abundance. In old mice, apigenin restored EDD (96±2%) by increasing NO bioavailability, normalized arterial ROS, oxidative stress and antioxidant expression, and abolished ROS inhibition of EDD. Moreover, apigenin prevented foam cell formation in vitro (initiating step in atherosclerosis) and mitigated age-associated aortic stiffening (PWV 373±5 cm/sec) by normalizing aortic intrinsic wall stiffness, collagen, elastin, AGEs, and inflammation. Thus, apigenin is a promising therapeutic for arterial aging.

Association of Aortic Stiffness With Biomarkers of Neuroinflammation, Synaptic Dysfunction, and Neurodegeneration

Objectives:To test the hypothesis that increased aortic stiffening is associated with greater cerebrospinal fluid (CSF) evidence of core Alzheimer’s disease pathology (Aβ, phosphorylated tau (p-tau)), neurodegeneration (total tau (t-tau)), synaptic dysfunction (neurogranin), neuroaxonal injury (neurofilament light (NFL)), and neuroinflammation (YKL-40, sTREM2), we analyzed pulse wave velocity (PWV) data and CSF data among older adults.Methods:Participants free of stroke and dementia from the Vanderbilt Memory and Aging Project, an observational community-based study, underwent cardiac magnetic resonance to assess aortic pulse wave velocity (PWV, m/sec) and lumbar puncture to obtain CSF. Linear regressions related aortic PWV to CSF Aβ, p-tau, t-tau, neurogranin, NFL, YKL-40, and sTREM2 concentrations adjusting for age, race/ethnicity, education, apolipoprotein (APOE) ε4 status, Framingham Stroke Risk Profile, and cognitive diagnosis. Models were repeated testing PWV interactions with age, diagnosis, APOE-ε4, and hypertension on each biomarker.Results:146 participants were examined (72±6 years). Aortic PWV interacted with age on p-tau (β=0.31, p=0.04), t-tau, (β=2.67, p=0.05), neurogranin (β=0.94, p=0.04), and sTREM2 (β=20.4, p=0.05). Among participants over age 73 years, higher aortic PWV related to higher p-tau (β=2.4, p=0.03), t-tau (β=19.3, p=0.05), neurogranin (β=8.4, p=0.01), and YKL-40 concentrations (β=7880, p=0.005). Aortic PWV had modest interactions with diagnosis on neurogranin (β=-10.76, p=0.03) and hypertension status on YKL-40 (β=-18020, p<0.001).Conclusions:Among our oldest participants, age 74 years and older, greater aortic stiffening is associated with in vivo biomarker evidence of neuroinflammation, tau phosphorylation, synaptic dysfunction, and neurodegeneration, but not amyloidosis. Central arterial stiffening may lead to cumulative cerebral microcirculatory damage and blood flow delivery to tissue, resulting in neuroinflammation and neurodegeneration in more advanced age.

Gut Microbiome-Derived Metabolite Trimethylamine N-Oxide Induces Aortic Stiffening and Increases Systolic Blood Pressure With Aging in Mice and Humans

Aging is associated with stiffening of the large elastic arteries and consequent increases in systolic blood pressure (SBP), which together increase cardiovascular disease risk; however, the upstream mechanisms are incompletely understood. Using complementary translational approaches in mice and humans, we investigated the role of the gut microbiome-derived metabolite trimethylamine N-oxide (TMAO) in age-related aortic stiffening and increased SBP. Aortic stiffness was measured using carotid-femoral or aortic pulse wave velocity (PWV) in humans and mice, respectively. Study 1: Plasma TMAO concentrations were elevated ( P <0.001) in healthy middle-aged to older (6.3±5.8 µmol/L) versus young (1.8±1.4 µmol/L) humans and positively related to carotid-femoral PWV ( r 2 =0.15, P <0.0001) and SBP ( r 2 =0.09, P <0.001), independent of traditional cardiovascular risk factors. Study 2: Dietary supplementation with TMAO increased aPWV in young mice and exacerbated the already elevated aPWV of old mice, accompanied by increases in SBP of ≈10 mm Hg in both groups. TMAO-supplemented versus control-fed mice also had higher intrinsic mechanical stiffness of the aorta (stress-strain testing) associated with higher aortic abundance of advanced glycation end-products, which form crosslinks between structural proteins to promote aortic stiffening. Study 3: Ex vivo incubation of aortic rings with TMAO increased intrinsic stiffness, which was attenuated by the advanced glycation end-products crosslink breaker alagebrium and prevented by inhibition of superoxide signaling. TMAO induces aortic stiffening and increases SBP via formation of advanced glycation end-products and superoxide-stimulated oxidative stress, which together increase intrinsic wall stiffness. Increases in circulating TMAO with aging represent a novel therapeutic target for reducing risk of aortic stiffening-related clinical disorders.