Aged Vessels Research Articles

Aged Lymphatic Vessels and Mast Cells in Perilymphatic Tissues.

This review provides a comprehensive summary of research on aging-associated alterations in lymphatic vessels and mast cells in perilymphatic tissues. Aging alters structure (by increasing the size of zones with low muscle cell investiture), ultrastructure (through loss of the glycocalyx), and proteome composition with a concomitant increase in permeability of aged lymphatic vessels. The contractile function of aged lymphatic vessels is depleted with the abolished role of nitric oxide and an increased role of lymphatic-born histamine in flow-dependent regulation of lymphatic phasic contractions and tone. In addition, aging induces oxidative stress in lymphatic vessels and facilitates the spread of pathogens from these vessels into perilymphatic tissues. Aging causes the basal activation of perilymphatic mast cells, which, in turn, restricts recruitment/activation of immune cells in perilymphatic tissues. This aging-associated basal activation of mast cells limits proper functioning of the mast cell/histamine/NF-κB axis that is essential for the regulation of lymphatic vessel transport and barrier functions as well as for both the interaction and trafficking of immune cells near and within lymphatic collecting vessels. Cumulatively, these changes play important roles in the pathogenesis of alterations in inflammation and immunity associated with aging.

Histamine as an Endothelium-Derived Relaxing Factor in Aged Mesenteric Lymphatic Vessels.

Knowledge of the mechanisms by which aging affects contracting lymphatic vessels remains incomplete; therefore, the functional role of histamine in the reaction of aged lymphatic vessels to increases in flow remains unknown. We measured and analyzed parameters of lymphatic contractility in isolated and pressurized rat mesenteric lymphatic vessels (MLVs) obtained from 9- and 24-month Fischer-344 rats under control conditions and after pharmacological blockade of nitric oxide (NO) by Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 100 μM) or/and blockade of histamine production by α-methyl-DL-histidine dihydrochloride (α-MHD, 10 μM). We also quantitatively compared results of immunohistochemical labeling of the histamine-producing enzyme, histidine decarboxylase (HDC) in adult and aged MLVs. Our data provide the first demonstration of an increased functional role of histamine as an endothelial-derived relaxing factor in aged MLVs, which appears in parallel with the abolished role of NO in the reactions of these lymph vessels to increases in flow. In addition, we found an increased expression of HDC in endothelium of aged MLVs. Our findings provide the basis for better understanding of the processes of aging in lymphatic vessels and for setting new important directions for investigations of the aging-associated disturbances in lymph flow and the immune response.

Complement Component C3 is a critical point in aged choroidal vessels in the complement factor H knock out model of retinal degeneration.

Complement Component C3 is a critical point in aged choroidal vessels in the complement factor H knock out model of retinal degeneration.

Mast cells and histamine are triggering the NF-κB-mediated reactions of adult and aged perilymphatic mesenteric tissues to acute inflammation.

This study aimed to establish mechanistic links between the aging-associated changes in the functional status of mast cells and the altered responses of mesenteric tissue and mesenteric lymphatic vessels (MLVs) to acute inflammation. We used an in vivo model of acute peritoneal inflammation induced by lipopolysaccharide treatment of adult (9-month) and aged (24-month) F-344 rats. We analyzed contractility of isolated MLVs, mast cell activation, activation of nuclear factor-κB (NF-κB) without and with stabilization of mast cells by cromolyn or blockade of all types of histamine receptors and production of 27 major pro-inflammatory cytokines in adult and aged perilymphatic mesenteric tissues and blood. We found that the reactivity of aged contracting lymphatic vessels to LPS-induced acute inflammation was abolished and that activated mast cells trigger NF-κB signaling in the mesentery through release of histamine. The aging-associated basal activation of mesenteric mast cells limits acute inflammatory NF-κB activation in aged mesentery. We conclude that proper functioning of the mast cell/histamine/NF-κB axis is necessary for reactions of the lymphatic vessels to acute inflammatory stimuli as well as for interaction and trafficking of immune cells near and within the collecting lymphatics.

Amyloid beta deposition and phosphorylated tau accumulation are key features in aged choroidal vessels in the complement factor H knock out model of retinal degeneration

Extra-cellular deposition including amyloid beta (Aβ) is a feature of retinal ageing. It has been documented for Bruch’s membrane (BM) where Aβ is elevated in complement factor H knockout mice (Cfh−/−) proposed as a model for age related macular degeneration. However, arterial deposition in choroidal vessels prior to perfusion across BM has not been examined. Aβ is associated with tau phosphorylation and these are linked in blood vessels in Alzheimers Disease where they can drive perivascular pathology. Here we ask if Aβ, tau and phosphorylated tau are features of ageing in choroidal vessels in 12 month C57 BL/6 and Cfh−/− mice, using immune staining and Western blot analysis.Greater levels of Aβ and phosphorylated tau are found in choroidal vessels in Cfh−/− mice. Western blot revealed a 40% increase in Aβ in Cfh−/− over C57 BL/6 mice. Aβ deposits coat around 55% of the luminal wall in Cfh−/− compared to only about 40% in C57 BL/6. Total tau was similar in both groups, but phosphorylated tau increased by >100% in Cfh−/− compared to C57 BL/6 and covered >75% of the luminal wall compared to 50% in C57 BL/6. Hence, phosphorylated tau is a marked choroidal feature in this mouse model. Aβ deposition was clumped in Cfh−/− mice and likely to influence blood flow dynamics. Disturbed flow is associated with atherogenesis and may be related to the accumulation of membrane attack complex recently identified between choroidal vessels in those at high risk of macular degeneration due to complement factor H polymorphisms.

Mast Cells‐Dependent Compromised Response of Aged Mesenteric Lymphatic Vessels and Surrounding Tissues to the Acute Inflammatory Challenge

Mesenteric lymphatic vessels (MLVs), being located at the border between the biologically aggressive environment of the gut lumen and inner compartments of the abdomen have not been studied in depth as an active component of the anti‐inflammatory defense system of the body. Aging creates several challenges for lymphatic vessels by altering their ultrastructure, contractility and consequently lymph flow as well as their microenvironment. We found an increase in number and degree of basal activation of mast cells (MC) nearby aged mesenteric lymphatic vessels. Such, as we predicted, will delimit ability of aged mesenteric tissue to respond to acute inflammatory challenge. In current study we attempted to establish mechanistic links between the aging‐associated changes in functional status of MCs and the altered responses of mesenteric tissue and MLVs to acute inflammatory insult. We used in vivo model of induction of acute peritoneal inflammation by lipopolysaccharide (LPS) treatment (24 hours after single IP injection of LPS in dose of 10 mg/kg body weight into adult (9‐mo) and aged (24‐mo) F‐344 rats). Analysis of data obtained by studies based on ruthenium red staining of MCs and by flow cytometry‐based measurements demonstrate that LPS‐induced degranulation of MCs in aged mesentery is greatly diminished. Latter co‐exists with basal activation of NF‐κB signaling in aged MLVs and mesenteric tissue therefore additional LPS‐induced NF‐κB activation is also reduced. Furthermore both mast cell stabilization by Cromolyn, or application of mixture of Histamine H1–H4 receptor blockers, are able to prevent the NF‐κB activation in mesentery/MLVs. NF‐κB, as hallmark of aging‐associated low‐grade inflammation, controls the release of pro‐inflammatory cytokines. To identify the corresponding aging‐associated differences, 24 hours after LPS injection we compared blood and mesenteric tissue (including MLVs) samples from adult and aged rats for 27 major pro‐inflammatory cytokines, using a Multiplex cytokine assay system (kit RECMAG65K27PMX, Millipore, US). The obtained results show that there is an elevation of NF‐κB‐dependent pro‐inflammatory cytokines in aged mesentery/MLVs with imbalance of Th1/Th2 cytokine profile and subsequent changes in T and B cells populations. Cumulatively, our data indicate that pre‐existing activation of MCs in aged mesentery by MLVs linked to its altered response to LPS, associated with histamine/NF‐κB‐dependent specific changes in cytokine levels. Latter can contribute to imbalance of immune cells profile and decreased ability of the aged immune system to respond to acute inflammatory challenge in peritoneal compartment of the body.Support or Funding InformationNIH/NIDDK 1R56DK099161

Aging-related anatomical and biochemical changes in lymphatic collectors impair lymph transport, fluid homeostasis, and pathogen clearance.

The role of lymphatic vessels is to transport fluid, soluble molecules, and immune cells to the draining lymph nodes. Here, we analyze how the aging process affects the functionality of the lymphatic collectors and the dynamics of lymph flow. Ultrastructural, biochemical, and proteomic analysis indicates a loss of matrix proteins, and smooth muscle cells in aged collectors resulting in a decrease in contraction frequency, systolic lymph flow velocity, and pumping activity, as measured in vivo in lymphatic collectors. Functionally, this impairment also translated into a reduced ability for in vivo bacterial transport as determined by time-lapse microscopy. Ultrastructural and proteomic analysis also indicates a decrease in the thickness of the endothelial cell glycocalyx and loss of gap junction proteins in aged lymph collectors. Redox proteomic analysis mapped an aging-related increase in the glycation and carboxylation of lymphatic’s endothelial cell and matrix proteins. Functionally, these modifications translate into apparent hyperpermeability of the lymphatics with pathogen escaping from the collectors into the surrounding tissue and a decreased ability to control tissue fluid homeostasis. Altogether, our data provide a mechanistic analysis of how the anatomical and biochemical changes, occurring in aged lymphatic vessels, compromise lymph flow, tissue fluid homeostasis, and pathogen transport.

Aging-associated chronic mast cell activation by aged mesenteric lymphatic vessels (INC6P.324)

Abstract Here we present novel data related to interactions between lymphatic vessels, mast cells (MCs), eosinophils, and immune cells. MCs are known to inhabit in greater density the tissue near mesenteric lymphatic vessels (MLVs). Acute activation induces MC degranulation with subsequent recruitment of eosinophils and of MHC class II+ cells towards MLVs. Aging permanently influences mesenteric MCs by inducing chronic basal activation, thereby influencing contractility of aged MLVs. At the same time, the number of intact MCs available to react acutely to inflammatory stimuli decreases with age. The diminished number and activity of MCs in aged tissues also diminishes traffic and activation of eosinophils that are attracted to aged MLVs. Concurrently, MCs can function as helper cells for B cells that can produce IgE. IgE has a relatively short half-life in the absence of chronic MC activation. We propose that a key component of aging-associated alterations of “mesenteric lymphatic system-mast cells/eosinophils-inflammatory cells” system is the increased basal activation of resident MCs in aged mesentery that promote chronic inflammation with lesser potential to recruit enough anti-inflammatory MHC class II+ cells and eosinophils towards MLVs, resulting in the inability to prevent a smooth transition from acute inflammation to specific immune cell activation.

Elastin aging and lipid oxidation products in human aorta

Vascular aging is associated with structural and functional modifications of the arteries, and by an increase in arterial wall thickening in the intima and the media, mainly resulting from structural modifications of the extracellular matrix (ECM) components. Among the factors known to accumulate with aging, advanced lipid peroxidation end products (ALEs) are a hallmark of oxidative stress-associated diseases such as atherosclerosis. Aldehydes generated from the peroxidation of polyunsaturated fatty acids (PUFA), (4-hydroxynonenal, malondialdehyde, acrolein), form adducts on cellular proteins, leading to a progressive protein dysfunction with consequences in the pathophysiology of vascular aging. The contribution of these aldehydes to ECM modification is not known. This study was carried out to investigate whether aldehyde-adducts are detected in the intima and media in human aorta, whether their level is increased in vascular aging, and whether elastin fibers are a target of aldehyde-adduct formation. Immunohistological and confocal immunofluorescence studies indicate that 4-HNE-histidine-adducts accumulate in an age-related manner in the intima, media and adventitia layers of human aortas, and are mainly expressed in smooth muscle cells. In contrast, even if the structure of elastin fiber is strongly altered in the aged vessels, our results show that elastin is not or very poorly modified by 4-HNE. These data indicate a complex role for lipid peroxidation and in particular for 4-HNE in elastin homeostasis, in the vascular wall remodeling during aging and atherosclerosis development.

Pulse Height Spectral Studies of Ozonizer Discharges in Argon at a Moderate Pressure and the Associated Joshi Effect

The effect of light has been observed in the variation of discharge counts with pulse amplitude during d.c.. discharges through argon in a fresh and an aged Siemen's vessels. The pulse height spectra of the discharge for applied potentials of 1.75 and 1.82 kV (r.m.s.) have been measured in dark and under irradiation. The number of current pulses varies periodically with increasing amplitude both in the low and plateau-like intermediate amplitude regions; finally the number of pulse drops rapidly to negligible value in the high amplitude regions. The theoretical explanation for the various observed features in the plots has been attempted on the properties of adsorbed layers on the glass walls of the discharge tube.

Abstract 336: The Imbalanced Redox Homeostasis in Senescent Endothelial Cells is Due to Dysregulated Thioredoxin-1 and NADPH-Oxidase 4

Senescent endothelial cells are not only present in aged vessels, but also in atherosclerotic lesions, where they contribute to endothelial dysfunction. One hallmark of cellular senescence is an imbalanced redox homeostasis. However, the underlying mechanisms how this imbalance occurs are not completely understood. Thioredoxin-1 (Trx-1) is one of the most important redox regulators in primary human endothelial cells. It has previously been shown that Trx-1, in addition to its oxidoreductase activity has anti-apoptotic properties. Cellular generators of reactive oxygen species (ROS) are NADPH oxidases (NOXs), of which NOX4 shows highest expression levels in endothelial cells. Therefore, the aim of the study was to investigate whether and how Trx-1 and NOX4 are regulated during stress-induced premature senescence in endothelial cells, an ex vivo model mirroring the situation in aged or diseased vessels. We treated primary human endothelial cells for two weeks with hydrogen peroxide to generate stress-induced premature senescence in these cells. Senescence induction was confirmed by an increase in senescence-associated beta-Galactosidase and nuclear p21, two established senescence markers. Moreover, cytosolic and mitochondrial reactive oxygen species formation were enhanced. Concomitantly, we observed decreased Trx-1 levels and elevated NOX4 levels, which could explain the imbalance in the cellular redox homeostasis. Moreover, the lysosomal protease Cathepsin D, known to degrade Trx-1 under short-term oxidative stress, was over-activated, providing a mechanistic basis for reduced Trx-1 protein levels. Inhibition of “over-active” Cathepsin D by the specific, cell-permeable inhibitor pepstatin A counteracted the increase in nuclear p21, ROS formation and degradation of Trx-1, thus leading to blockade of stress-induced premature senscence by stabilizing the cellular redox homeostasis. Moreover, aortic Trx-1 levels negatively correlated with NOX4 expression in NOX4 transgenic mice exclusively expressing NOX4 in the endothelium and their wildtype littermates. Our data show that loss of Trx-1 and upregulation of NOX4 may importantly contribute to the imbalanced redox homeostasis in senescent endothelial cells ex vivo and in vivo.

Functional consequences of chronic mast cell activation by aged mesenteric lymphatic vessels (666.1)

Here we present novel data related to interactions between lymphatic vessels, mast cells (MCs), eosinophils, and immune cells. MCs are known to inhabit in greater density the tissue near mesenteric lymphatic vessels (MLVs). Acute activation induces MC degranulation with subsequent recruitment of eosinophils and of MHC class II+ cells towards MLVs. Aging permanently influences mesenteric MCs by inducing chronic basal activation, thereby influencing contractility of aged MLVs. At the same time, the number of intact MCs available to react acutely to inflammatory stimuli decreases with age. The diminished number and activity of MCs in aged tissues also diminishes traffic and activation of eosinophils and other immune cells that are attracted to aged MLVs. Concurrently, MCs can function as helper cells for B cells that can produce IgE. IgE has a relatively short half‐life in the absence of chronic MC activation. We propose that a key component of aging‐associated alterations of “mesenteric lymphatic system‐mast cells/eosinophils‐inflammatory cells” system is the increased basal activation of resident MCs and eosinophils in aged mesentery that promote chronic inflammation while being insufficient to recruit enough anti‐inflammatory MHC class II+ cells and eosinophils, resulting in the inability to prevent a smooth transition from acute inflammation to specific immune cell activation. NIH R01 AG030578.Grant Funding Source: NIH R01 AG030578

Korean red ginseng water extract restores impaired endothelial function by inhibiting arginase activity in aged mice.

Cardiovascular disease is the prime cause of morbidity and mortality and the population ages that may contribute to increase in the occurrence of cardiovascular disease. Arginase upregulation is associated with impaired endothelial function in aged vascular system and thus may contribute to cardiovascular disease. According to recent research, Korean Red Ginseng water extract (KRGE) may reduce cardiovascular disease risk by improving vascular system health. The purpose of this study was to examine mechanisms contributing to age-related vascular endothelial dysfunction and to determine whether KRGE improves these functions in aged mice. Young (10±3 weeks) and aged (55±5 weeks) male mice (C57BL/6J) were orally administered 0, 10, or 20 mg/mouse/day of KRGE for 4 weeks. Animals were sacrificed and the aortas were removed. Endothelial arginase activity, nitric oxide (NO) generation and reactive oxygen species (ROS) production, endothelial nitric oxide synthase (eNOS) coupling, vascular tension, and plasma peroxynitrite production were measured. KRGE attenuated arginase activity, restored nitric oxide (NO) generation, reduced ROS production, and enhanced eNOS coupling in aged mice. KRGE also improved vascular tension in aged vessels, as indicated by increased acetylcholine-induced vasorelaxation and improved phenylephrine-stimulated vasoconstriction. Furthermore, KRGE prevented plasma peroxynitrite formation in aged mice, indicating reduced lipid peroxidation. These results suggest KRGE exerts vasoprotective effects by inhibiting arginase activity and augmenting NO signaling and may be a useful treatment for age-dependent vascular diseases.

Muscle cells in rat mesenteric lymphatic vessels of various ages

The detailed quantitative evaluation of potential aging‐associated changes in muscle cell density in mesenteric lymphatic vessels (MLV) has never been performed. In this study we, for the first time, performed detailed evaluation of muscle cell density in MLV in reference to the position of lymphatic valve in different zones of lymphangion within various age groups (3‐mo, 9‐mo and 24‐mo Fischer‐344 rats). Using visual and quantitative analyses of the images of MLV immunohistochemically labeled for actin, we confirmed that the zones located close upstream and above lymphatic valves possess the lowest density of lymphatic muscle cells. Most of the high muscle cell density zones exist downstream to the lymphatic valve. The muscle cell density of these zones is not affected by aging while pre‐valve and valve zones demonstrate significant aging‐associated decrease in muscle cell density. The low muscle cell density zones in lymphatic vessels consist of predominantly longitudinally oriented muscle cells which are positioned above and near lymphatic valve and connect adjacent lymphangions. These cells may provide important functional impact on the biomechanics of the lymphatic valve gating and electrical coupling between lymphangions, while their aging‐associated changes may delimit adaptive reserves of aged lymphatic vessels. NIH R01 AG030578, HL094269.

Lymphatic Muscle Cells in Rat Mesenteric Lymphatic Vessels of Various Ages

Recent studies on aging-associated changes in mesenteric lymph flow in situ demonstrated predominance of the severe negative chronotropic effect of aging on the contractility of aged mesenteric lymphatic vessels (MLV). At the same time, contraction amplitude of the aged vessels was only slightly diminished by aging and can be rapidly stimulated within 5-15 minutes. However, the detailed quantitative evaluation of potential aging-associated changes in muscle cells investiture in MLV has never been performed. In this study we, for the first time, performed detailed evaluation of muscle cells investiture in MLV in reference to the position of lymphatic valve in different zones of lymphangion within various age groups (3-mo, 9-mo and 24-mo Fischer-344 rats). Using visual and quantitative analyses of the images of MLV immunohistochemically labeled for actin, we confirmed that the zones located close upstream (pre-valve zones) and above lymphatic valves (valve zones) possess the lowest investiture of lymphatic muscle cells. Most of the high muscle cells investiture zones exist downstream to the lymphatic valve (post-valve zones). The muscle cells investiture of these zones is not affected by aging, while pre-valve and valve zones demonstrate significant aging-associated decrease in muscle cells investiture. The low muscle cells investiture zones in lymphatic vessels consist of predominantly longitudinally oriented muscle cells which are positioned in pre-valve and valve zones and connect adjacent lymphangions. These cells may provide important functional impact on the biomechanics of the lymphatic valve gating and electrical coupling between lymphangions, while their aging-associated changes may delimit adaptive reserves of aged lymphatic vessels.

Aged Lymphatic Contractility: Recent Answers and New Questions

Abstract An overview is presented of recent findings related to biology of aging of the lymph transport system. The authors discuss recently obtained data on the aging-associated alterations of lymphatic contractility in thoracic duct and mesenteric lymphatic vessels; on comparisons of function of aged mesenteric lymphatic vessels in situ versus isolated specimens and important conclusions which arose from these studies; on aging-associated changes in functional status of mast cells located close to aged mesenteric lymphatic vessels; on evidence of presence of oxidative stress in aged lymphatic vessels and changes in arrangement of muscle cells in their walls. The authors conclude that future continuation of the research efforts in this area is necessary and will be able to provide not only novel fundamental knowledge on the biology of lymphatic aging, but also will create solid foundation for the subsequent developments of lymphatic-oriented therapeutic interventions in many diseases of the elderly.

IMPAIRED VASCULAR RESPONSES TO CALCITONIN GENE-RELATED PEPTIDE IN THE AGEING α-CGRP KNOCKOUT MOUSE

Calcitonin gene-related peptide (CGRP) is a potent microvascular vasodilator derived predominantly from perivascular sensory neurones. It has been suggested that vascular reactivity to CGRP is diminished with increasing age whilst...

Age-associated alterations in retinal arteriole reactivity to endothelin-1 differ between the sexes

Endothelin-1 (ET-1) is a vasoconstrictor implicated in age-related retinal pathologies. This study determined whether responses to ET-1 differed in retinal arterioles isolated from adult (2–3 months) and aged (>20 months) Fischer 344 rats of both sexes. Risk factors for retinal disease (retinal perfusion pressure, intraocular pressure, blood glucose) were not affected by age. However, sensitivity to ET-1 declined with age, especially in females. Vasoconstrictor responses to 50mM KCl and Ca2+ release by caffeine (10mM) were similar in all groups. Retinal ETA and ETB receptor expression also was similar in young and aged rats, regardless of sex. Contractions elicited by 10nM ET-1 were inhibited by the ETA antagonist BQ-123 (1μM) in all groups. In contrast, the ETB antagonist BQ-788 (1μM) restored ET-1-induced contractions in aged female vessels, but had no effect in any other group. Removal of the endothelium also restored contractions in vessels from aged females but not males. Thus, responsiveness to ET-1 declines with age in retinal microvasculature. In males, this is likely mediated by age-related changes in the ETA receptor signaling pathway. By contrast, effects of ET-1 on endothelial ETB receptors attenuate vasoconstrictor responses in aged females.

Abstract 120: Notch Signaling Negatively Regulates Vascular Endothelial Cell Senescence

Accumulation of senescent vascular cells occurs in aged vessels, which leads to an increase of inflammation and a decline of regenerative potential, thereby promoting vascular dysfunction and atherosclerosis. However, the molecular mechanisms of these age-related changes remain unclear. The Notch pathway is a highly conserved signaling that controls cell fate determination and differentiation during the development of various tissues. In adults, Notch signaling has been reported to be essential for neovascularization and is implicated with the age-associated conditions such as cancer, neuronal disorder and impaired regeneration of skeletal muscle. Here, we show that Notch signaling has a crucial role in endothelial cell senescence. We found that inhibition of Notch signaling, using short hairpin RNA (shRNA) targeting Notch1, reduced the maximum population doublings of human umbilical vein endothelial cells (HUVEC), increased the activity of senescence-associated beta-galactosidase, and up-regulated the expression of aging-associated molecules such as p53, p21, and p16. Knockdown of the Notch ligand Jagged1 resulted in attenuation of Notch signaling, thereby inducing premature senescence in a way similar to knockdown of Notch1. In contrast, over-expression of Notch1 or Jagged1 extended the replicative lifespan of HUVEC and decreased the expression of aging-associated molecules. To elucidate the mechanism how inhibition of Notch signaling induces premature senescence in endothelial cells, we examined expression of various molecules by microarray analysis, and found that the expression of Id1 (inhibitor of DNA binding 1) and MKP1 (MAP kinase phosphatase 1) was significantly down-regulated by knockdown of Notch. Over-expression of Id1 improved Notch1 inhibition-induced premature senescence, which is associated with a decrease of p16 expression. Likewise, treatment with SB203580, an inhibitor of p38 MAPK, extended lifespan of Notch-deleted endothelial cells along with down-regulation of p16. These results suggest that Notch signaling and the downstream molecules (Id1 and p38) regulate endothelial cell senescence presumably via a p16-dependent pathway, and may be a new target of the therapy for age-associated vascular diseases.

Aging-associated shifts in functional status of mast cells located by adult and aged mesenteric lymphatic vessels

We had previously proposed the presence of permanent stimulatory influences in the tissue microenvironment surrounding the aged mesenteric lymphatic vessels (MLV), which influence aged lymphatic function. In this study, we performed immunohistochemical labeling of proteins known to be present in mast cells (mast cell tryptase, c-kit, prostaglandin D(2) synthase, histidine decarboxylase, histamine, transmembrane protein 16A, and TNF-α) with double verification of mast cells in the same segment of rat mesentery containing MLV by labeling with Alexa Fluor 488-conjugated avidin followed by toluidine blue staining. Additionally, we evaluated the aging-associated changes in the number of mast cells located by MLV and in their functional status by inducing mast cell activation by various activators (substance P; anti-rat DNP Immunoglobulin E; peptidoglycan from Staphyloccus aureus and compound 48/80) in the presence of ruthenium red followed by subsequent staining by toluidine blue. We found that there was a 27% aging-associated increase in the total number of mast cells, with an ∼400% increase in the number of activated mast cells in aged mesenteric tissue in resting conditions with diminished ability of mast cells to be newly activated in the presence of inflammatory or chemical stimuli. We conclude that higher degree of preactivation of mast cells in aged mesenteric tissue is important for development of aging-associated impairment of function of mesenteric lymphatic vessels. The limited number of intact aged mast cells located close to the mesenteric lymphatic compartments to react to the presence of acute stimuli may be considered contributory to the aging-associated deteriorations in immune response.