Effects of aging on single cardiac myocyte function in Fischer 344 x Brown Norway rats

The Fischer 344 x Brown Norway (F344xBN) rat has been demonstrated to have a lower incidence of age-related pathology than other rat strains. Therefore, to elucidate the effects of aging on cardiac function, uncomplicated by compensatory effects caused by age-related pathology, cardiac myocytes were isolated from female F344xBN rats at 6 (young) and 32-33 (old) mo of age. Myocytes showed an increase in the relative amount of beta-myosin heavy chain with advanced age and a significant rightward shift in the tension-pCa curve from 5.78 +/- 0.02 pCa units in young adult myocytes to 5.66 +/- 0.03 pCa units. Consistent with a shift to a slower myosin isoform, the time from stimulation to peak sarcomere shortening increased with age from 50.5 +/- 1.3 to 58.9 +/- 1.0 ms. In contrast, no age-related difference was found in either the relengthening parameters or the Ca(2+) transient, indicating that relaxation is not directly altered by aging. This latter finding is at variance with previous studies in rat strains with higher rates of pathology. We conclude, therefore, that the primary effect of aging in isolated cardiac myocytes from the F344xBN rat model is a shift in the myosin heavy chain isoform. Changes in relaxation seen in other rat strains may result from compensatory mechanisms induced by pathological conditions.     

Impact of genetic background and aging on mesenteric collateral growth capacity in Fischer 344, Brown Norway, and Fischer 344 x Brown Norway hybrid rats

Available studies indicate that both genetic background and aging influence collateral growth capacity, but it is not known how their combination affects collateral growth. We evaluated collateral growth induced by ileal artery ligation in Fischer 344 (F344), Brown Norway (BN), and the first generation hybrid of F344 x BN (F1) rats available for aging research from the National Institute on Aging. Collateral growth was determined by paired diameter measurements in anesthetized rats immediately and 7 days postligation. In 3-mo-old rats, significant collateral growth occurred only in BN (35% +/- 11%, P < 0.001). The endothelial cell number in arterial cross sections was also determined, since this precedes shear-mediated luminal expansion. When compared with the same animal controls, the intimal cell number was increased only in BN rats (92% +/- 21%, P < 0.001). The increase in intimal cell number and the degree of collateral luminal expansion in BN rats was not affected by age from 3 to 24 mo. Immunohistochemical studies demonstrated that intimal cell proliferation was much greater in the collaterals of BN than of F1 rats. The remarkable difference between these three strains of rats used in aging research and the lack of an age-related impairment in the BN rats are novel observations. These rat strains mimic clinical observations of interindividual variation in collateral growth capacity and the impact of age on arteriogenesis and should be useful models to investigate the molecular mechanisms responsible for such differences.     

Upregulation of G protein-linked receptor kinases with advancing age in rat aorta

The age-related decline in beta-adrenergic receptor (beta-AR)-mediated vasorelaxation is associated with desensitization of beta-ARs without significant downregulation. The primary mode of this homologous beta-AR desensitization, in general, is via G protein receptor kinases (GRK). Therefore, we hypothesize that age-related changes in GRKs are causative to this etiology in rat aorta. Herein, we investigate the activity and cellular distribution (cytoplasmic vs. membrane) of several GRK isoforms and beta-arrestin proteins. GRK activity was assessed in extracts from aortic tissue of 6-wk, 6-mo, 12-mo, and 24-mo-old male Fischer-344 rats using a rhodopsin phosphorylation assay. We also performed immunoblots on lysates from aorta with specific antibodies to GRK-2, -3, -5, and beta-arrestin-1. Results show an age-related increase in GRK activity. Furthermore, expression of GRK-2 (cytoplasmic and membrane), GRK-3 (cytoplasmic and membrane), and beta-arrestin (soluble) increased with advancing age, whereas GRK-5 (membrane) expression remained unchanged. These results suggest that age is associated with increased activity and expression of specific GRKs. This increase likely results in enhanced phosphorylation and desensitization of beta-ARs. These biochemical changes are consistent with observed aging physiology.     

Relationships among element contents in the intimal, middle, and external tunicae of the thoracic aorta

To examine an accumulation of elements within the arteries with aging, the authors investigated the element contents in the intimal, middle, and external tunicae of the thoracic aorta. The subjects consisted of six men and four women, ranging in age from 57 to 99 yr. The wall of the thoracic aorta was separated into the intimal, middle, and external tunicae by scrubbing the wall of the thoracic aorta with an edge of slide glass and the element contents were determined by inductively coupled plasma-atomic emission spectrometry. It was found that there were significant relationships among calcium, phosphorus, magnesium, sulfur, and sodium in both the intimal and middle tunicae of the aorta, but not in the external tunica. These results revealed that no significant differences were found in element compositions of deposits between the intimal and middle tunicae.     

Aging‐related carcinoembryonic antigen‐related cell adhesion molecule 1 signaling promotes vascular dysfunction

Aging is an independent risk factor for cardiovascular diseases and therefore of particular interest for the prevention of cardiovascular events. However, the mechanisms underlying vascular aging are not well understood. Since carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1) is crucially involved in vascular homeostasis, we sought to identify the role of CEACAM1 in vascular aging. Using human internal thoracic artery and murine aorta, we show that CEACAM1 is upregulated in the course of vascular aging. Further analyses demonstrated that TNF‐α is CEACAM1‐dependently upregulated in the aging vasculature. Vice versa, TNF‐α induces CEACAM1 expression. This results in a feed‐forward loop in the aging vasculature that maintains a chronic pro‐inflammatory milieu. Furthermore, we demonstrate that age‐associated vascular alterations, that is, increased oxidative stress and vascular fibrosis, due to increased medial collagen deposition crucially depend on the presence of CEACAM1. Additionally, age‐dependent upregulation of vascular CEACAM1 expression contributes to endothelial barrier impairment, putatively via increased VEGF/VEGFR‐2 signaling. Consequently, aging‐related upregulation of vascular CEACAM1 expression results in endothelial dysfunction that may promote atherosclerotic plaque formation in the presence of additional risk factors. Our data suggest that CEACAM1 might represent an attractive target in order to delay physiological aging and therefore the transition to vascular disorders such as atherosclerosis.     

Vitamin C increases the prostacyclin production and decreases the vascular lesions in experimental atherosclerosis in rabbits

Feeding a cholesterol-rich diet (0.3%) to rabbits resulted in an intimal thickening and lipid infiltration of the aorta. The prostacyclin production by the vascular endothelium was significantly decreased, after a transient increase after 2 weeks of diet. The arachidonic acid metabolism in platelets was hardly changed. Addition of a low dose vitamin C (150 mg/day) to the cholesterol rich diet resulted in decreased lipid infiltration and intimal thickening and the transient increase of the prostacyclin production was postponed to the 4th week. Although this dose of vitamin C could not restore the decreased prostacyclin production observed after 6 weeks diet, a higher dose of vitamin C (600 mg/day), besides its beneficial effect on the lipid infiltration and the intimal thickening in the thoracic aorta, kept the intimal prostacyclin production at normal levels for at least 8 weeks.     

Neuroimmunology of healthy brain aging

Aging involves progressive deterioration away from homeostasis. Whereas the healthy adult brain maintains neuroimmune cells in a surveillant and homeostatic state, aged glial cells have a hyperreactive phenotype. These age-related pro-inflammatory biases are driven in part by cell-intrinsic factors, including increased cell priming and pro-inflammatory cell states. In addition, the aged inflammatory milieu is shaped by an altered environment, such as amplified danger signals and cytokines and dysregulated glymphatic function. These cell-instrinsic and environmental factors conspire to heighten the age-related risk for neuroimmune activation and associated pathology. In this review, we discuss cellular and molecular neuroimmune shifts with “healthy” aging; how these age-related changes affect physiology and behavior; and how recent research has revealed neuroimmune pathways and targets for improving health span.     

Potential Contribution of Phenotypically Modulated Smooth Muscle Cells and Related Inflammation in the Development of Experimental Obstructive Pulmonary Vasculopathy in Rats

We tested the hypothesis that phenotypically modulated smooth muscle cells (SMCs) and related inflammation are associated with the progression of experimental occlusive pulmonary vascular disease (PVD). Occlusive PVD was induced by combined exposure to a vascular endothelial growth factor receptor tyrosine kinase inhibitor Sugen 5416 and hypobaric hypoxia for 3 weeks in rats, which were then returned to ambient air. Hemodynamic, morphometric, and immunohistochemical studies, as well as gene expression analyses, were performed at 3, 5, 8, and 13 weeks after the initial treatment (n = 78). Experimental animals developed pulmonary hypertension and right ventricular hypertrophy, and exhibited a progressive increase in indices of PVD, including cellular intimal thickening and intimal fibrosis. Cellular intimal lesions comprised α smooth muscle actin (α SMA)+, SM1+, SM2+/-, vimentin+ immature SMCs that were covered by endothelial monolayers, while fibrous intimal lesions typically included α SMA+, SM1+, SM2+, vimentin+/- mature SMCs. Plexiform lesions comprised α SMA+, vimentin+, SM1-, SM2- myofibroblasts covered by endothelial monolayers. Immature SMC-rich intimal and plexiform lesions were proliferative and were infiltrated by macrophages, while fibrous intimal lesions were characterized by lower proliferative abilities and were infiltrated by few macrophages. Compared with controls, the number of perivascular macrophages was already higher at 3 weeks and progressively increased during the experimental period; gene expression of pulmonary hypertension-related inflammatory molecules, including IL6, MCP1, MMP9, cathepsin-S, and RANTES, was persistently or progressively up-regulated in lungs of experimental animals. We concluded that phenotypically modulated SMCs and related inflammation are potentially associated with the progression of experimental obstructive PVD.     

Altered proteasome function and subunit composition in aged muscle

Myofibrillar protein degradation is mediated through the ubiquitin-proteasome pathway. To investigate if altered proteasome activity plays a role in age-related muscle atrophy, we examined muscle size and proteasome function in young and aged F344BN rats. Significant age-related muscle atrophy was confirmed by the 38% decrease in cross-sectional area of type 1 fibers in soleus muscle. Determination of proteasome function showed hydrolysis of fluorogenic peptides was equivalent between ages. However, when accounting for the 3-fold increase in content of the 20S catalytic core in aged muscle, the lower specific activity suggests a functional loss in individual proteins with aging. Comparing the composition of the catalytic beta-subunits showed an age-related 4-fold increase in the cytokine-inducible subunits, LMP2 and LMP7. Additionally, the content of the activating complexes, PA28 and PA700, relative to the 20S proteasome was reduced 50%. These results suggest significant alterations in the intrinsic activity, the percentage of immunoproteasome, and the regulation of the 20S proteasome by PA28 and PA700 in aged muscle.     

Age-related nephropathy in the Fischer 344 rat is associated with overexpression of collagens and collagen-binding heat shock protein 47

To explore the possible role of heat shock protein (HSP) 47 in the age-related renal changes in Fischer 344 (F 344) rats, the expression of collagen-binding HSP47 with various proteins implicated in phenotypic modulation (α-smooth muscle actin, desmin, and vimentin) and fibrosis (type I, type III, and type IV collagens) was examined in young and old F 344 rat kidneys. Male F 344 rats often develop spontaneous nephropathy in old age. Kidneys obtained from 24-month-old F 344 rats showed glomerulosclerosis with marked tubulointerstitial damage including interstitial fibrosis, while no significant histological alteration was found in the kidneys of 6-month-old rats. Immunohistochemical analysis showed an increased accumulation of type I, type III, and type IV collagens in areas of glomerulosclerosis and interstitial fibrosis in old rat kidneys. In kidneys of young rats, collagen-binding HSP47 expression was weak in the glomeruli and occasionally seen in the interstitial cells. In contrast, strong immunostaining for HSP47 was noted in the glomeruli, tubular epithelial cells, and interstitial cells in kidneys of old rats. In addition, phenotypic alterations of mesangial cells and interstitial cells (immunopositive for α-smooth muscle actin), glomerular epithelial cells (immunopositive for desmin), and tubular epithelial cells (immunopositive for vimentin) were found in the kidneys of old F 344 rats. Double immunostaining showed that all these phenotypically altered renal cells express HSP47 and that increased expression of HSP47 was always associated with increased expression of collagens in the old rat kidneys. From the above observations, it is concluded that overexpression of HSP47 by phenotypically altered renal cells might play an important role in the excessive assembly of collagens and could thereby contribute to the glomerulosclerosis and interstitial fibrosis found in kidneys of aged F 344 rats.     

Age-related differences in insulin-like growth factor-1 receptor signaling regulates Akt/FOXO3a and ERK/Fos pathways in vascular smooth muscle cells

Advanced age is a major risk factor for atherosclerosis, but how aging per se influences pathogenesis is not clear. Insulin-like growth factor-1 receptor (IGF-1R) promotes aortic vascular smooth muscle cell (VSMC) growth, migration, and extracellular matrix formation, but how IGF-1R signaling changes with age in VSMC is not known. We previously found age-related differences in the activation of Akt/FOXO3a and ERK1/2 pathways in VSMC, but the upstream signaling remains unclear. Using explanted VSMC from Fischer 344/Brown Norway F1 hybrid rats shown to display age-related vascular pathology similar to humans, we compared IGF-1R expression in early passages of VSMC and found a constitutive activation of IGF-1R in VSMC from old compared to young rats, including IGF-1R expression and its tyrosine kinase activity. The link between IGF-1R activation and the Akt/FOXO3a and ERK pathways was confirmed through the induction of IGF-1R with IGF-1 in young cells and attenuation of IGF-1R with an inhibitor in old cells. The effects of three kinase inhibitors: AG1024, LY294002, and TCN, were compared in VSMC from old rats to differentiate IGF-1R from other upstream signaling that could also regulate the Akt/FOXO and ERK pathways. Genes for p27kip-1, catalase and MnSOD, which play important roles in the control of cell cycle arrest and stress resistance, were found to be FOXO3a-targets based on FOXO3a-siRNA treatment. Furthermore, IGF-1R signaling modulated these genes through activation of the Akt/FOXO3a pathway. Therefore, activation of IGF-1R signaling influences VSMC function in old rats and may contribute to the increased risk for atherosclerosis.     

Trophic factor supplemented UW solution reduces intimal hyperplasia in the rat aortic transplant model

BACKGROUND: Chronic allograft nephropathy (CAN) is associated with delayed graft function, cold ischemic injury, and is an important cause of premature graft loss. A characteristic vascular lesion of CAN is intimal hyperplasia (IH). The goal of this study was to evaluate the effect of cold storage in University of Wisconsin solution supplemented with trophic factors (UW-TF) on IH in rat aortic isograft (IG) and allograft (AG) models. METHODS: F344 --> F344 and Lewis --> F344 orthotopic abdominal aortic transplants were performed after 48 h of cold storage in either UW or UW-TF solution with and without immunosuppression. RESULTS: Significant reduction in IH was observed when IG were stored in UW-TF solution compared to UW solution. A significant reduction in intimal inflammation was observed in UW-TF stored, nonimmunosuppressed AG. In immunosuppressed recipients, AG stored in UW-TF solution evidenced significantly less IH compared to those stored in UW alone. CONCLUSIONS: UW-TF solution decreased IH in both alloindependent and dependent models.     

Characteristics of the aortic elastic network and related phenotypes in seven inbred rat strains

Extracellular matrix (ECM) molecules such as elastin and collagen provide mechanical support to the vessel wall and are essential for vascular function. Evidence that genetic factors influence aortic ECM composition and organization was concluded from our previous studies showing that the inbred Brown Norway (BN) rat differs significantly from the outbred Long-Evans (LE) and the inbred LOU rat with respect to both thoracic aortic elastin content and internal elastic lamina (IEL) rupture in the abdominal aorta and iliac arteries. Here, we measured aortic elastin and collagen contents as well as factors that may modulate these parameters [insulin growth factor (IGF)-I, transforming growth factor (TGF)-beta(1), and matrix metalloproteinase (MMP)-2] in seven inbred rat strains, including BN and LOU. We also investigated whether IEL ruptures occur in strains other than BN. We showed that LOU, LE, BN, and Fischer 344 (F344) rats were significantly different for aortic elastin content and elastin-to-collagen ratio, whereas LE, Lewis, WAG, and Wistar-Furth (WF) were similar for these parameters. BN and F344 had the lowest values. BN was the only strain to present numerous IEL ruptures, whereas F344, LE, and WF presented a few and the other strains presented none. In addition, IGF-I and TGF-beta(1) levels in the plasma and aorta differed significantly between strains, suggesting genetic control of their production. Because inbred rat strains provide interesting models for quantitative trait locus analysis, our results concerning elastin, collagen, IEL ruptures, and cytokines may provide a basis for the search for candidate genes involved in the control of these phenotypes.     

Chronic 1alpha,25-(OH)2 vitamin D3 treatment reduces Ca2+ -mediated hippocampal biomarkers of aging

Aging in the hippocampus of several species is characterized by alterations in multiple Ca(2+)-mediated processes, including an increase in L-type voltage-gated Ca(2+) channel (L-VGCC) current, an enhanced Ca(2+)-dependent slow afterhyperpolarization (AHP), impaired synaptic plasticity and elevated Ca(2+) transients. Previously, we found that 1alpha,25-dihydoxyvitamin D(3) (1,25VitD), a major Ca(2+) regulating hormone, down-regulates L-VGCC expression in cultured hippocampal neurons. Here, we tested whether in vivo treatment of aged F344 rats with 1,25VitD would reverse some of the Ca(2+) -mediated biomarkers of aging seen in hippocampal CA1 neurons. As previously reported, L-VGCC currents and the AHP were larger in aged than in young neurons. Treatment with 1,25VitD over 7 days decreased L-VGCC activity in aged rats, as well as the age-related increase in AHP amplitude and duration. In addition, reduced L-VGCC activity was correlated with reduced AHPs in the same animals. These data provide direct evidence that 1,25VitD can regulate multiple Ca(2+)-dependent processes in neurons, with particular impact on reducing age-related changes associated with Ca(2+) dysregulation. Thus, these results may have therapeutic implications and suggest that 1,25VitD, often taken to maintain bone health, may also retard some consequences of brain aging.     

Compromised mechanical homeostasis in arterial aging and associated cardiovascular consequences

Aging leads to central artery stiffening and associated hemodynamic sequelae. Because healthy arteries exhibit differential geometry, composition, and mechanical behaviors along the central vasculature, we sought to determine whether wall structure and mechanical function differ across five vascular regions—the ascending and descending thoracic aorta, suprarenal and infrarenal abdominal aorta, and common carotid artery—in 20 versus 100-week-old male wild-type mice. Notwithstanding generally consistent changes across these regions, including a marked thickening of the arterial wall, diminished in vivo axial stretch, and loss of elastic energy storage capacity, the degree of changes tended to be slightly greater in abdominal than in thoracic or carotid vessels. Likely due to the long half-life of vascular elastin, most mechanical changes in the arterial wall resulted largely from a distributed increase in collagen, including thicker fibers in the media, and localized increases in glycosaminoglycans. Changes within the central arteries associated with significant increases in central pulse pressure and adverse changes in the left ventricle, including increased cardiac mass and decreased diastolic function. Given the similar half-life of vascular elastin in mice and humans but very different life-spans, there are important differences in the aging of central vessels across these species. Nevertheless, the common finding of aberrant matrix remodeling contributing to a compromised mechanical homeostasis suggests that studies of central artery aging in the mouse can provide insight into mechanisms and treatment strategies for the many adverse effects of vascular aging in humans.     

Effects of Ginkgo biloba extract on the proliferation of vascular smooth muscle cells in vitro and on intimal thickening and interleukin-1beta expression after balloon injury in cholesterol-fed rabbits in vivo

Restenosis may develop in response to cytokine activation and smooth muscle cell proliferation. Ginkgo biloba extract (EGb) has been used to treat cardiovascular and cerebrovascular diseases. In the present study, the effects of EGb on the growth of cultured vascular smooth muscle cells (VSMC), as well as on the expression of interleukin-1beta (IL-1beta) and the intimal response in balloon-injured arteries of cholesterol-fed rabbits, were investigated. Using bromodeoxyuridine incorporation as an index of cell proliferation, EGb was found to inhibit serum-induced mitogenesis of cultured rat aorta VSMC in a dose-dependent manner. In vivo, EGb and probucol ( positive control) reduced the atheroma area in thoracic aortas of male New Zealand white rabbits fed a 2% cholesterol diet for 6 weeks with balloon denudation of the abdominal aorta being performed at the end of the third week. Intimal hyperplasia, expressed as the intimal/medial area ratio, in the abdominal aortas was significantly inhibited in the both the EGb group (0.61 +/- 0.06) and the probucol group (0.55 +/- 0.03) compared to the C group (0.87 +/- 0.02). In the balloon-injured abdominal aorta, both EGb and probucol significantly reduced IL-1beta mRNA and protein expression and the percentage of proliferating cells. The inhibitory effects of EGb on the intimal response might be attributed to its antioxidant capacity. EGb may have therapeutic potential for the prevention of restenosis after angioplasty.     

Stress,Aging, and Brain Oxidative Damage

Stress may contribute to aging acceleration and age-related degenerative diseases. Stress and adaptation to stress require numerous homeostatic adjustments including hormones, neurotransmitters, oxidants, and other mediators. The stress-induced hormones, neurotransmitters, and oxidants all have beneficial, but also harmful effects if out of balance. Therefore, the homeostasis of stress and adaptation should be governed by the hormone balance, neurotransmitter balance, and oxidant balance, as well as the interactions among these substances. The imbalance and the over-interaction of these balances may ultimately cause increased oxidant generation and oxidative damage to biomolecules. This increased oxidative damage may add to the oxidant burden associated with normal aerobic metabolism, which in itself, generates oxidants, causes accumulation of oxidative damage in mitochondria, and contributes to normal aging. Therefore, the stress-associated increase of oxidative damage may, in part, contribute to stress-associated aging acceleration and age-related neurodegenerative diseases.