Young Aorta Research Articles

Humoral and cellular factors inhibit phosphate-induced vascular calcification during the growth period.

Hyperphosphatemia is an independent and non-classical risk factor of cardiovascular disease and mortality in patients with chronic kidney disease (CKD). Increased levels of extracellular inorganic phosphate (Pi) are known to directly induce vascular calcification, but the detailed underlying mechanism has not been clarified. Although serum Pi levels during the growth period are as high as those observed in hyperphosphatemia in adult CKD, vascular calcification does not usually occur during growth. Here, we have examined whether the defence system against Pi-induced vascular calcification can exist during the growth period using mice model. We found that calcification propensity of young serum (aged 3 weeks) was significantly lower than that of adult serum (10 months), possibly due to high fetuin-A levels. In addition, when the aorta was cultured in high Pi medium in vitro, obvious calcification was observed in the adult aorta but not in the young aorta. Furthermore, culture in high Pi medium increased the mRNA level of tissue-nonspecific alkaline phosphatase (TNAP), which degrades pyrophosphate, only in the adult aorta. Collectively, our findings indicate that the aorta in growing mouse may be resistant to Pi-induced vascular calcification via a mechanism in which high serum fetuin-A levels and suppressed TNAP expression.

Differential endothelial senescence elicited by AQP1 regulation of epigenetic/metabolic responses

Abstract Introduction Accumulation of senescent endothelial cells with age is an important driver of vascular aging and related cardiovascular disease. However, little is known about the mechanisms and signaling pathways that underpin endothelial cell senescence. Aquaporin-1 (AQP1) has been implicated in sensing hydrogen peroxide (H2O2), which differentially modulates endothelial signal transduction. Nonetheless, the role of AQP1 in the regulation of metabolism and function in young and old vascular cells remains elusive. Purpose We sought to investigate whether 1) AQP1 differentially regulates vascular senescence and angiogenesis through epigenetic/metabolic response, senescence-associated secretory phenotype (SASP), and 2) AQP1 knockdown/inhibition rejuvenates senescent endothelial cells and restores angiogenesis. Methods We assessed the role of AQP1 in endothelial cell senescence and impaired angiogenesis through mitochondrial metabolic disorder, epigenetic alteration, and the SASP in human aortic endothelial cells and aortas from wild-type C57BL/6 mice. For these, we conducted AQP1 knockdown (with siRNA) or blockade of AQP1 (with Bacopaside-II, 10 μM) to inhibit H2O2 transport. Results Here we show the upregulation of AQP1 in the senescent endothelial cells (SEC, p.15) and aorta from old mice (>24 months) (p<0.01 and p<0.0001, respectively), facilitating outside-in H2O2 transport. Interestingly, AQP1 knockdown/inhibition reverses both replicative (increased SA-β-galactosidase positive cells) and stress-induced senescence (increased cell-cycle arrest markers p16, p19, and p21; and SASP markers IL1α, IL1β, and IL6) in SEC, whereas it triggers the senescence processes in the proliferating endothelial cells (PEC, p.5) (for each, p<0.05, n=6). Unlike in PEC or young aorta (3-month-old), AQP1 knockdown/inhibition restores angiogenesis in both SEC and old aortas. The interventions increased mitochondrial OXPHOS (represented by enhanced oxygen consumption rate (OCR)) in SEC, which is accompanied by marked increase in CaMKKβ-regulated AMPKT172 phosphorylation (for each, p<0.05, n=6) and in histone deacetylase-4 (HDAC4) phosphorylation at Ser632 (p<0.05) and further histone 3 (H3) acetylation (p<0.01). The HDAC4 phosphorylation leads to significant increase in Mef2A-mediated eNOS phosphorylation at Ser1177 (p<0.05). By contrast, AQP1 knockdown/inhibition induced a senescent-like state by a decrease in mitochondrial OCR and the phosphorylation of CaMKKβ-AMPK and HDAC4-Mef2A regulation of eNOS activity. Conclusion Our studies identify AQP1 as essential regulator of EC proliferation that differentially regulates angiogenesis via epigenetic/metabolic response and SASP modulation. Moreover, genetic or pharmacological inhibition of AQP1 reverses endothelial senescence and restores angiogenesis, representing a potential therapeutic target for vascular rejuvenation. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Swiss National Science Foundation (SNSF)SwissLife Jubiläumsstiftung

Aging Alters the Aortic Proteome in Health and Thoracic Aortic Aneurysm.

Aging enhances most chronic diseases but its impact on human aortic tissue in health and in thoracic aortic aneurysms (TAA) remains unclear. We employed a human aortic biorepository of healthy specimens (n=17) and those that underwent surgical repair for TAA (n=20). First, we performed proteomics comparing aortas of healthy donors to aneurysmal specimens, in young (ie, <60 years of age) and old (ie, ≥60 years of age) subjects. Second, we measured proteins, via immunoblotting, involved in mitophagy (ie, Parkin) and also mitochondrial-induced inflammatory pathways, specifically TLR (toll-like receptor) 9, STING (stimulator of interferon genes), and IFN (interferon)-β. Proteomics revealed that aging transformed the aorta both quantitatively and qualitatively from health to TAA. Whereas young aortas exhibited an enrichment of immunologic processes, older aortas exhibited an enrichment of metabolic processes. Immunoblotting revealed that the expression of Parkin directly correlated to subject age in health but inversely to subject age in TAA. In TAA, but not in health, phosphorylation of STING and the expression of IFN-β was impacted by aging regardless of whether subjects had bicuspid or tricuspid valves. In subjects with bicuspid valves and TAAs, TLR9 expression positively correlated with subject age. Interestingly, whereas phosphorylation of STING was inversely correlated with subject age, IFN-β positively correlated with subject age. Aging transforms the human aortic proteome from health to TAA, leading to a differential regulation of biological processes. Our results suggest that the development of therapies to mitigate vascular diseases including TAA may need to be modified depending on subject age.

Abstract 8929: Mineralocorticoid Receptor as an Epigenetic Regulator of Vascular Aging

Introduction: Vascular stiffness increases with age and independently predicts cardiovascular events but vascular fibrosis mechanisms are unclear. Epigenetic changes accumulate with age and contribute to aging pathology. We showed that aging-associated increase in vascular stiffness/fibrosis are mitigated by treating old mice with mineralocorticoid receptor (MR) antagonist (MRA) or by genetic deletion of MR in smooth muscle cells (SMC-MR-KO). Hypothesis: We hypothesized that SMC-MR regulates vascular fibrosis genes by modulating epigenetic histone modifications in the aging vasculature. Methods and Results: Global histone proteomic profiling revealed increased acetylation (ac) and decreased methylation (me) of histone H3 lysine 27 (H3K27) in old vs young mouse aorta that is attenuated by SMC-MR-KO. Similarly, RNA profiling shows downregulation of fibrotic genes (CTGF, MMP2 and Integrin-α5) with aging in mouse aortas from SMC-MR-KO mice. Mechanism was explored in primary low passage human aortic SMC (HASMC). Comparison of protein levels in HASMCs from old vs young people shows increased MR, decreased H3K27-specific methyltransferase EZH2, increased H3K27ac and increased fibrosis gene expression in aged HASMC. Treatment of young HASMC with H 2 O 2 (aging inductor) increased MR and decreased EZH2 in association with decreased H3K27me. These epigenetic changes were associated with increased H3K27ac and MR enrichment at the promoter of fibrosis genes by ChIP along with increased fibrosis gene expression. Conversely, treatment of aged HASMC with MRA increased EZH2 and H3K27me with decreased fibrosis gene expression. Finally, treatment of 12 month old mice with MR antagonist for 4 months or deletion of MR from SMC prevented aging induction of vascular stiffness by pulse wave velocity in association with decreased H3K27ac at promoters and decreased expression of fibrosis genes in aging aortas. Conclusion: These data support a model in which rising SMC-MR with aging contributes to vascular fibrosis via EZH2 reduction leading to decreased H3K27me and increased H3K27ac and MR recruitment to promote transcription of fibrotic genes. These findings suggest the therapeutic potential for MRAs for aging-associated vascular stiffening/fibrosis.

The role of transient receptor potential ankyrin 1 in age-related endothelial dysfunction.

Oxidative stress plays a key role in age-related vascular disease. The present study aimed to investigate the role of an antioxidant channel, transient receptor potential ankyrin 1 (TRPA1), in age-related endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) were grown to induce replicative senescence, and 6-month-old young, 12-month-old middle-aged, and 24-month-old aged mice were used. TRPA1 was downregulated in senescent HUVECs, so were endothelial nitric oxide synthase (eNOS), nuclear factor erythroid 2-related factor 2 (Nrf2), and uncoupling protein 2 (UCP2). Activating TRPA1 with cinnamaldehyde prevented downregulation of eNOS, Nrf2, and UCP2, inhibited superoxide production and apoptosis, and preserved nitric oxide bioavailability in senescent HUVECs. TRPA1, phosphorylated eNOS, Nrf2 and UCP2 were significantly downregulated in aged aortas compared with young aortas after a compensatory upregulation in middle-aged aortas. Dietary administration of cinnamaldehyde for 12months prevented mitochondrial dysfunction, improved endothelium-dependent relaxation, and increased expression of eNOS, Nrf2, and UCP2 in aged aortas. Importantly, the effects of cinnamaldehyde can be blocked by a TRPA1 antagonist HC-030031. These findings suggest that TRPA1 may play a critical role in age-related endothelial dysfunction and may become a therapeutic target for the treatment and prevention of age-related vascular disease.

Abstract P281: Vascular Remodeling And Impaired Vascular Smooth Muscle Cell Plasticity In Age And Sex-dependent Hypertension

Aim: Hypertension (HTN) and aging are associated with the development of vascular dysfunction. We speculated that vascular smooth muscle cell plasticity and vascular remodeling play major roles in age and sex-dependent HTN. Methods: Male and female Sprague-Dawley (SD) rats aged 3 and 16-months-old (N=6/group) were housed under standard conditions. Blood pressure was measured via femoral artery cannulation and sympathetic tone to the vasculature was estimated by ganglion blockade via Hexamethonium (30mg/Kg IV). PBS-perfused abdominal aorta and renal arteries were collected and immunoblotting was performed following protein extraction. Results: Male SD rats, but not females, develop HTN and increased sympathetic tone with age. Aged hypertensive male rats, but not aged normotensive females, exhibit reduced p-Erk1/2 and p-eNos levels in both abdominal aorta and renal arteries. α-Smooth Muscle Actin significantly increased in aged male abdominal aorta. Elevated c-Src was observed in aged female abdominal aorta and p-c-Src was reduced in aged male abdominal aorta. Caveolin-1 changed oppositely in young and aged abdominal aorta and renal arteries of two sexes. Conclusions: Our data suggest that artery-specific changes of key signaling molecules contribute to impaired vascular smooth muscle plasticity and vascular dysfunction in aged hypertensive male but not in aged normotensive female rats.

Microstructural and mechanical characterization of the layers of human descending thoracic aortas

The mechanical properties of human aortas are linked to the layered tissue and its microstructure at different length scales. Each layer has specific mechanical and structural properties. While the ground substance and the elastin play an important role in tissue stiffness at small strain, collagen fibers carry most of the load at larger strains, which corresponds to the physiological conditions of the aorta at maximum pulsatile blood pressure. In fact, collagen fibers are crimped in the unloaded state. Collagen fibers show different orientation distributions when they are observed in a plane that is tangent to the aortic wall (in-plane section) or along a direction orthogonal to it (out-of-plane section). This was systematically investigated using large images (2500 × 2500 µm) with high resolution obtained by second harmonic generation (SHG) in order to homogenize tissue heterogeneity after a convergence analysis, which is a main goal of the study. In addition, collagen fibers show lateral interactions due to entanglements and the presence of transverse elastin fibers, observed on varying length scales using atomic force microscopy and a three-dimensional rendering obtained by stacking a sequence of SHG and two-photon fluorescence images; this is another important contribution. Human descending thoracic aortas from 13 heartbeat donors aged 28 to 66 years were examined. Uniaxial tensile tests were carried out on the longitudinal and circumferential strips of the aortic wall and the three separated layers (intima, media and adventitia). A structurally-motivated material model with (i) a term to describe the combined response of ground substance and elastin and (ii) terms to consider four families of collagen fibers with different directions was applied. The exclusion of compressed fibers was implemented in the fitting process of the experimental data, which was optimized by a genetic algorithm. The results show that a single fiber family with directional and dispersion parameters measured from SHG images can describe the mechanical response of all 39 layers (3 layers for each of the 13 aortas) with very good accuracy when a second (auxiliary) family of aligned fibers is introduced in the orthogonal direction to account for lateral fiber interaction. Indeed, all observed distributions of collagen directions can be accurately fitted by a single bivariate von Mises distribution. Statistical analysis of in-plane and out-of-plane dispersion of fiber orientations reveals structural differences between the three layers and a change of collagen dispersion parameters with age. Statement of significanceThe stiffness of healthy young aortas is adjusted so that a diameter expansion of about 10 % is possible during the heartbeat. This creates the Windkessel effect, which smooths out the pulsating nature of blood flow and benefits organ perfusion. The specific elastic properties of the aorta that are required to achieve this effect are related to the microstructure of the aortic tissue at different length scales. An increase in the aortic stiffness, in addition to reducing cyclic expansion and worsening perfusion, is a risk factor for clinical hypertension. The present study relates the microstructure of healthy human aortas to the mechanical response and examines the changes in microstructural parameters with age, which is a key factor in increasing stiffness.

The correlation of IRE1α oxidation with Nox4 activation in aging-associated vascular dysfunction

Oxidative stress attributable to the activation of a Nox4-containing NADPH oxidase is involved in aging-associated vascular dysfunction. However, the Nox4-induced signaling mechanism for the vascular alteration in aging remains unclear. In an aged aorta, the expression of Nox4 mRNA and protein by Nox family of genes was markedly increased compared with a young aorta. Nox4 localization mainly to ER was also established. In the aorta of Nox4 WT mice aged 23–24 months (aged), reactive oxygen species (ROS) and endoplasmic reticulum (ER)/oxidative stress were markedly increased compared with the counter KO mice. Furthermore, endothelial functions including eNOS coupling process and acetylcholine-induced vasodilation were significantly disturbed in the aged WT, slightly affected in the counter KO aorta. Consistently, in d-galactose-induced in vitro aging condition, ER-ROS and its associated ER Nox4 expression and activity were highly increased. Also, in chronic d-galactose-treated condition, IRE1α phosphorylation and XBP-1 splicing and were transiently increased, but IRE1α sulfonation was robustly increased in the aging Nox4 WT condition when compared to the counter KO condition. In vitro D-gal-induced aging study, the phenomenon were abrogated with Nox4 knock-down condition and was significantly decreased in GKT, Nox4 inhibitor and 4-PBA, ER chemical chaperone-treated human umbilical vein endothelial cells. The state of Nox4-based ER redox imbalance/ROS accumulation is suggested to determine the pathway “the UPR; IRE1α phosphorylation and XBP-1 splicing and the UPR failure; IRE1α cysteine-based oxidation, especially sulfonation, finally controlling aging-associated vascular dysfunction.

Evaluation of the Effects of Aging on the Aorta Stiffness in Relation with Mineral and Trace Element Levels: an Optimized Method via Custom-Built Stretcher Device.

Aortic stiffness represents the major cause of aging and tightly associated with hypertension, atherosclerosis, cardiovascular diseases, and increased mortality. Mechanical characteristics of the aorta play a vital role in the blood flow, circulation, systolic pressure, and aortic stiffness; however, the correlation of trace element and mineral levels with aortic stiffness has not been studied before. Balance in the trace elements and minerals is vital for the biological functions; however, natural aging may alter this balance. Thus, after measuring aortic stiffness of aged and young rat aortas by a custom-built stretcher device, trace element and mineral levels were evaluated via ICP-MS. Also, biomarkers of aging including blood pressure, arterial pressure glucose, insulin levels, and histochemical parameters were investigated as well. Aortic stiffness, blood glucose, plasma insulin, systolic, diastolic, and mean arterial pressure significantly increased by aging in the aorta of aged rats compared with the young ones. Also, Fe, Al, Co, Ni, Zn, Sr, Na, Mg, and K levels increased in the aged aorta samples compared with the young aorta samples of rats. Increased levels of the indicated elements may be correlated with the development and progression of aortic stiffness and vascular complications. Thus, possible mechanisms correlating aortic stiffness with the imbalance in the trace element and mineral levels should be further investigated.

Effects of longitudinal pre-stretch on the mechanics of human aorta before and after thoracic endovascular aortic repair (TEVAR) in trauma patients.

Thoracic endovascular aortic repair (TEVAR) has evolved as a first-line therapy for trauma patients. Most trauma patients are young, and their aortas are compliant and longitudinally pre-stretched. We have developed a method to include longitudinal pre-stretch in computational models of human thoracic aortas of different ages before and after TEVAR. Finite element models were built using computerized tomography angiography data obtained from human subjects in 6 age groups 10-69years old. Aortic properties were determined with planar biaxial testing, and pre-stretch was simulated using a series of springs. GORE C-Tag stent-graft was computationally deployed in aortas with and without pre-stretch, and the stress-strain fields were compared. Pre-stretch had significant qualitative and quantitative effects on the aortic stress-strain state before and after TEVAR. Before TEVAR, mean intramural aortic stresses with and without pre-stretch decreased with age from 108kPa and 83kPa in the youngest age group, to 60kPa in the oldest age group. TEVAR increased intramural stresses by an average of 73 ± 15kPa and 48 ± 10kPa for aortas with and without pre-stretch and produced high stress concentrations near the aortic isthmus. Inclusion of pre-stretch in young aortas increased intramural stresses by 30%, while in > 50-year-old subjects it did not change the results. Computational modeling of aorta-stent-graft interaction that includes pre-stretch can be instrumental for device design and assessment of its long-term performance, and in the future may help more accurately determine the stress-strain characteristics associated with TEVAR complications.

The matrix proteins aggrecan and fibulin-1 play a key role in determining aortic stiffness

Stiffening of the aorta is an important independent risk factor for myocardial infarction and stroke. Yet its genetics is complex and little is known about its molecular drivers. We have identified for the first time, tagSNPs in the genes for extracellular matrix proteins, aggrecan and fibulin-1, that modulate stiffness in young healthy adults. We confirmed SNP associations with ex vivo stiffness measurements and expression studies in human donor aortic tissues. Both aggrecan and fibulin-1 were found in the aortic wall, but with marked differences in the distribution and glycosylation of aggrecan reflecting loss of chondroitin-sulphate binding domains. These differences were age-dependent but the striking finding was the acceleration of this process in stiff versus elastic young aortas. These findings suggest that aggrecan and fibulin-1 have critical roles in determining the biomechanics of the aorta and their modification with age could underpin age-related aortic stiffening.

Ageing of the vascular endothelium: a novel role for CaMKIIδ

Calcium/calmodulin–dependent protein kinase II delta (CaMKIIδ) plays a pivotal role in cardiovascular health and disease. Whether CaMKIIδ modulates impaired function in ageing cardiovasculature is unknown. This study investigates cardiac and aortic CaMKIIδ expression and activation in aged rats. Cardiovascular function was assessed in vivo by echocardiography. Reduced fractional shortening (63.2±1.3 vs 50.6±1.9 (%FS), young vs aged (n=12), p<0.01) and increased heart weight:body weight (2.7±0.2 vs 3.6±0.1, young vs aged (n=6), p<0.05) were observed in aged animals suggesting cardiac remodelling. Increased blood flow through the ascending aorta was also observed in aged rats (76.7±3.1 vs 103.4±7.4 (ml/min), young vs. aged (n=12), p<0.05). Parallel investigation of CaMKIIδ in cardiac and aortic tissue revealed increased protein expression (1.03±0.1 vs11.7±0.2, (n=7) p<0.05 (cardiac); 1.1±0.1 vs. 1.3±0.1 (aortic), young vs. aged (n=6) (CaMKIIδ/GAPDH)). Further analysis of aortic tissue showed increased activation of CaMKII with elevations in phosphorylated and oxidised CaMKII (ox-CaMKII) expression (1.2±0.1 vs 1.8±0.2 (n=5) p<0.05; 1.0±0.1 vs 1.3±0.08 (n=5) p<0.05, young vs. aged) as well as increased kinase activity (5.9±1.2 vs 8.2±1.4 (pmolPO4-inc/min/μg protein) young vs.aged, (n=3)). To explore these novel observations in the vasculature further, endothelial cells from young and aged aortae were isolated. These cells displayed striking phenotypic differences between groups with aged cells displaying clear signs of necrosis. To investigate whether ox-CaMKII may contribute to deterioration of the endothelium in ageing, initial experiments studied young endothelial cells exposed to H2O2 (10 µM). Reactive oxygen species and ox-CaMKII expression were measured in parallel and a corresponding increase in both parameters occurred following treatment (2.5 × 103 vs 4.4 × 104 (fluorescence a.u.) control vs treated, (n=3), p<0.01; 1.1±0.2 vs 1.82±0.1 (ox-CaMKII/GAPDH), control vs treated), p<0.001, (n=3)). This work provides new evidence that CaMKIIδ expression and activation are augmented in aged vasculature. Future work will examine CaMKII in aged endothelial cells to provide mechanistic insight into the deterioration of vascular function.

Age‐associated vascular inflammation promotes monocytosis during atherogenesis

SummaryAging leads to a proinflammatory state within the vasculature without disease, yet whether this inflammatory state occurs during atherogenesis remains unclear. Here, we examined how aging impacts atherosclerosis using Ldlr −/− mice, an established murine model of atherosclerosis. We found that aged atherosclerotic Ldlr −/− mice exhibited enhanced atherogenesis within the aorta. Aging also led to increased LDL levels, elevated blood pressure on a low‐fat diet, and insulin resistance after a high‐fat diet (HFD). On a HFD, aging increased a monocytosis in the peripheral blood and enhanced macrophage accumulation within the aorta. When we conducted bone marrow transplant experiments, we found that stromal factors contributed to age‐enhanced atherosclerosis. To delineate these stromal factors, we determined that the vasculature exhibited an age‐enhanced inflammatory response consisting of elevated production of CCL‐2, osteopontin, and IL‐6 during atherogenesis. In addition, in vitro cultures showed that aging enhanced the production of osteopontin by vascular smooth muscle cells. Functionally, aged atherosclerotic aortas displayed higher monocyte chemotaxis than young aortas. Hence, our study has revealed that aging induces metabolic dysfunction and enhances vascular inflammation to promote a peripheral monocytosis and macrophage accumulation within the atherosclerotic aorta.

177 Pro-inflammatory signalling in vascular endothelium: a novel role for camkii in ageing?

Nuclear factor-kappa beta (NF-kB) pro-inflammatory signalling is important in modulating endothelial dysfunction and may be important in vascular dysfunction associated with the ageing process. Recent studies in the heart have highlighted Calcium/calmodulin-dependent protein kinase II (CaMKII) as a novel regulator of NF-kB signalling. However nothing is known of the role this interaction could play in regulating dysfunction of the vasculature during ageing. Here we (i) characterise NF-kB signalling in vascular endothelial cells and examine the potential for CaMKII modulation and (ii) determine whether CaMKIIδ expression is altered in ageing. Using human umbilical vein endothelial cells (HUVECs) as an in vitro model system, initial experiments have established that pro-inflammatory NF- kB signalling is activated in response to both tumour necrosis factor α (TNF-α) and interleukin-1β (IL-1β) stimulation. This was shown by a significant reduction in IkBα expression (1.18±0.16 vs. 0.48±0.13, control vs. TNF-α 15 minutes stimulation ,p=0.008, n=3); (1.53±0.22 vs. 0.16±0.09 control vs. IL-1β 15 minutes stimulation ,p=0.004, n=3) and increased phosphorylation of the p65 subunit (0.69±0.1 vs. 1.22±0.12 ,control vs. TNF-α 5 minutes stimulation, p=0.006, n=3); (0.54±0.07 vs. 1.86±0.21, control vs. IL-1β 5 minutes stimulation ,p=0.005, n=3). We have identified CaMKIIδ as the predominant isoform expressed in HUVECs and shown CaMKII is also activated in response to TNF-α (1.3-fold increase at 10 minutes stimulation, p=0.01, n=3) and IL-1β. Further work has assessed expression of CaMKIIδ in freshly isolated rat aorta. Tissue lysate preparations have compared CaMKIIδ expression in young and aged aortae. Parallel studies in hearts from the same animals have shown CaMKIIδ expression is increased in whole ventricular homogenates prepared from aged versus young hearts (2.3-fold increase, n=3). These results reflect similar alterations in CaMKIIδ aged hearts as previously observed in models of cardiovascular disease. This highlights the possibility that CaMKII could be pivotal in modulating cardiovascular changes that occur during ageing. Future work will involve selective inhibition of CaMKIIδ in the HUVEC model system by siRNA transfection prior to cytokine stimulation. This will establish involvement of CaMKIIδ in modulation of NF-kB signalling in a vascular endothelial cell line. Studies can then be performed in primary aortic endothelial cells from both young and aged animals to further investigate this interaction and the potential importance in modulating endothelial dysfunction during ageing.

Abstract 48: Age-associated Imbalance of Vasorin/TGF-β1 Signaling in VSMC Facilitates Collagen Production

Collagen deposition, a hallmark of arterial aging that resembles post-injury arterial restenosis, is perpetrated by angiotensin II (Ang II) signaling in arterial wall. Collagen aggregation at sites of arterial injury is regulated by the coordinated signaling of pro-fibrotic TGF-β1 and anti-fibrotic vasorin within VSMCs. The Ang II/TGF-β1/vasorin signaling relationship within VSMCs with aging, however, remains unknown. In vivo studies in old vs. young FXBN rats show that aortic transcription and translation of vasorin markedly decrease with aging. In vitro studies in VSMCs isolated from old vs. young aortae. Ang II-associated reduction of vasorin protein abundance in young VSMCs and age-associated changes in vasorin protein levels are reversed by the AT1 antagonist, Losartan (Los) (Figure). Dual immunolabeling and co-immunoprecipitation demonstrate that the co-incidence and physical interaction of vasorin and TGF-β1 within VSMCs are significantly decreased with aging. Importantly, exposure of young VSMCs to Ang II that increases p-SMAD2/3 and collagen type I production, mimicking old cells, and this effect is abolished or substantially mitigated by Los treatment, overexpression of ectopic vasorin, or exogenous recombinant human-vasorin protein. In contrast, exposure of old VSMCs to Los decreases p-SMAD2/3 and collagen type I production.Thus, an imbalance of the Ang II/TGF-β1/vasorin signaling cascade, a feature of the aged arterial wall, enhances the collagen production by VSMCs. Maintaining this signaling balance is a novel measure to retard adverse extracellular matrix remodeling, a determinant of arterial stiffening with aging. (MW and GP co-first authors)

Differential expressions of microRNA between young and senescent endothelial cells

To explore the differential expressions of microRNA (miRNA) between young and senescent endothelial cells. Young and senescent aorta endothelial cells (EC) were isolated and cultured in young and old male C57BL/6J mice. Immunostaining of VIII factor was performed to identify the endothelial cells. The method of diphenyl tetrazolium bromide (MTT) was employed to compare the cell growth. Microarray was used to detect the differential expression of microRNA between young and senescent endothelial cells and the microarray results were confirmed by real-time polymerase chain reaction (PCR). The expression of endothelial nitric oxide synthase (eNOS) was detected by Western blot. Primarily cultured endothelial cells were confirmed by the VIII immunostaining factor. Senescent ECs grew more rapidly than young ECs in lower serum ex vivo. Excluding gender difference, miR-135a, miR-182, miR-96, miR-31, miR-126-3p and miR-362-5p were up-regulated over 2 folds in young ECs, and miR-335-3p and miR-335-5p up-regulated over 2 folds in senescent ECs by miRNA microarray and RT-PCR. The up-regulation of miR335-3p in old ECs and the up-regulation of miR-135a, miR-96 in the young ECs might contribute to a lower expression of eNOS in senescent ECs. The expression of miRNAs changes with advancing age and may result in differential expressions of downstream genes.

Increased Endothelial Exocytosis and Generation of Endothelin-1 Contributes to Constriction of Aged Arteries

Circulating levels of endothelin (ET)-1 and endogenous ET(A)-mediated constriction are increased in human aging. The mechanisms responsible are not known. Investigate the storage, release, and activity of ET-1 system in arteries from young and aged Fischer-344 rats. After NO synthase inhibition (L-NAME), thrombin contracted aged arteries, which was inhibited by endothelial denudation, ET(A) receptor antagonism (BQ123), and ECE inhibition (phosphoramidon, SM19712) or by inhibiting exocytosis (TAT-NSF, N-ethylmaleimide-sensitive factor inhibitor). Thrombin did not cause endothelium-dependent contraction of young arteries. In aged but not young arteries, thrombin rapidly increased ET-1 release, which was abolished by endothelium denudation or TAT-NSF. L-NAME did not affect ET-1 release. ET-1 immunofluorescent staining was punctate and distinct from von Willebrand factor (VWF). VWF and ET-1 immunofluorescent intensity was similar in young and aged quiescent arteries. Thrombin rapidly increased ET-1 staining and decreased VWF staining in aged but had no effect in young aortas. After L-NAME, thrombin decreased VWF staining in young aortas. NO donor DEA-NONOate (1 to 100 nmol/L) reversed thrombin-induced exocytosis in young (VWF) but not aged L-NAME-treated aortas (VWF, ET-1). Expression of preproET-1 mRNA and ECE-1 mRNA were increased in aged compared to young endothelium. BigET-1 levels and contraction to exogenous BigET-1 (but not ET-1) were also increased in aged compared to young arteries. The stimulated exocytotic release of ET-1 is dramatically increased in aged endothelium. This reflects increased reactivity of exocytosis, increased expression and storage of ET-1 precursor peptides, and increased expression of ECE-1. Altered endothelial exocytosis of ET-1 and other mediators may contribute to cardiovascular pathology in aging.

Milk Fat Globule Protein Epidermal Growth Factor-8

Advancing age induces aortic wall thickening that results from the concerted effects of numerous signaling proteins, many of which have yet to be identified. To search for novel proteins associated with aortic wall thickening, we have performed a comprehensive quantitative proteomic study to analyze aortic proteins from young (8 months) and old (30 months) rats and identified 50 proteins that significantly change in abundance with aging. One novel protein, the milk fat globule protein epidermal growth factor 8 (MFG-E8), increases 2.3-fold in abundance in old aorta. Transcription and translation analysis demonstrated that aortic MFG-E8 mRNA and protein levels increase with aging in several mammalian species including humans. Dual immunolabeling shows that MFG-E8 colocalizes with both angiotensin II and monocyte chemoattractant protein (MCP)-1 within vascular smooth muscle cells (VSMCs) of the thickened aged aortic wall. Exposure of early passage VSMCs from young aorta to angiotensin II markedly increases MFG-E8 and enhances invasive capacity to levels observed in VSMCs from old rats. Treatment of VSMCs with MFG-E8 increases MCP-1 expression and VSMCs invasion that are inhibited by the MCP-1 receptor blocker vCCI. Silencing MFG-E8 RNA substantially reduces MFG-E8 expression and VSMCs invasion capacity. The data indicate that arterial MFG-E8 significantly increases with aging and is a pivotal relay element within the angiotensin II/MCP-1/VSMC invasion signaling cascade. Thus, targeting of MFG-E8 within this signaling axis pathway is a potential novel therapy for the prevention and treatment of the age-associated vascular diseases such as atherosclerosis.

Exocytosis of Endothelin‐1 from Endothelial Weibel‐Palade Bodies Contributes to Vasoconstriction in Aged Arteries

Endothelin‐1 (ET) has been localized to Weibel‐Palade Bodies (WPB) in endothelial cells. Experiments were performed to analyze the role of WPB‐derived ET in arterial regulation. Aortas from young (3 months) and aged F344 rats (19 months) were isolated and processed for isometric tension recording, ET release and immunofluorescent analysis. After inhibition of NO synthase (L‐NAME, 100 µM), thrombin (1U/ml) caused rapid contraction of aged aortas that was abolished by endothelial‐denudation, the ETA receptor antagonist BQ123 (1 µM) or by TAT‐NSF (1 µM), a cell‐permeable inhibitor of N‐ethylmaleimide sensitive factor (NSF) and inhibitor of WPB exocytosis. In young aortas, thrombin (1 U/ml) evoked a small contraction that was not inhibited by BQ123 or endothelial denudation. The response to thrombin in aged aortas was associated with a 5‐fold increase in ET release that was prevented by TAT‐NSF or endothelial‐denudation. WPBs were visualized by immunofluorescent imaging of von Willebrand factor using laser scanning microscopy of aortic segments. In aged aortas, thrombin caused a marked translocation of WPB to the cell surface. In conclusion, the acute release and exocytosis of ET from endothelial cells can contribute to vascular regulation. The dramatic increase in this activity in aged arteries might contribute to thrombogenic and inflammatory events associated with aging. (NIH grants # HL 80119 and OH 8531)

Impaired shear stress-induced nitric oxide production through decreased NOS phosphorylation contributes to age-related vascular stiffness

Endothelial dysfunction and increased arterial stiffness contribute to multiple vascular diseases and are hallmarks of cardiovascular aging. To investigate the effects of aging on shear stress-induced endothelial nitric oxide (NO) signaling and aortic stiffness, we studied young (3-4 mo) and old (22-24 mo) rats in vivo and in vitro. Old rat aorta demonstrated impaired vasorelaxation to acetylcholine and sphingosine 1-phosphate, while responses to sodium nitroprusside were similar to those in young aorta. In a customized flow chamber, aortic sections preincubated with the NO-sensitive dye, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, were subjected to steady-state flow with shear stress increase from 0.4 to 6.4 dyn/cm(2). In young aorta, this shear step amplified 4-amino-5-methylamino-2',7'-difluorofluorescein fluorescence rate by 70.6 +/- 13.9%, while the old aorta response was significantly attenuated (23.6 +/- 11.3%, P < 0.05). Endothelial NO synthase (eNOS) inhibition, by N(G)-monomethyl-l-arginine, abolished any fluorescence rate increase. Furthermore, impaired NO production was associated with a significant reduction of the phosphorylated-Akt-to-total-Akt ratio in aged aorta (P < 0.05). Correspondingly, the phosphorylated-to-total-eNOS ratio in aged aortic endothelium was markedly lower than in young endothelium (P < 0.001). Lastly, pulse wave velocity, an in vivo measure of vascular stiffness, in old rats (5.99 +/- 0.191 m/s) and in N(omega)-nitro-l-arginine methyl ester-treated rats (4.96 +/- 0.118 m/s) was significantly greater than that in young rats (3.64 +/- 0.068 m/s, P < 0.001). Similarly, eNOS-knockout mice demonstrated higher pulse wave velocity than wild-type mice (P < 0.001). Thus impaired Akt-dependent NO synthase activation is a potential mechanism for decreased NO bioavailability and endothelial dysfunction, which likely contributes to age-associated vascular stiffness.