This review focuses on molecular, cellular, and functional changes that occur in the vasculature during aging; explores the links between mitochondrial oxidative stress, inflammation, and development of vascular disease in the elderly patients; and provides a landscape of molecular mechanisms involved in cellular oxidative stress resistance, which could be targeted for the prevention or amelioration of unsuccessful vascular aging. Practical interventions for prevention of age-associated vascular dysfunction and disease in old age are considered here based on emerging knowledge of the effects of anti-inflammatory treatments, regular exercise, dietary interventions, and caloric restriction mimetics.

For a long time, locally released metabolic factors from the parenchyma and the myogenic response were considered to be the two main local regulatory mechanisms to control microvascular resistance. However, the mediation of many responses, such as reactive and functional hyperemia could not be satisfactorily explained by these two mechanisms. The idea that an increase in blood flow may itself cause vessel dilation evolved at the beginning of the last century, although earlier observations have been documented. In the last two decades, it has been established that increases in blood flow velocity in the presence of a constant diameter lead to increases in wall shear stress, which is followed by vasodilatation. It has also been shown that endothelial mechanisms are involved in the transduction of the shear stress signal. More importantly, the role of shear stress mechanisms has been recognized to be essential in the regulation of basal vascular tone of microvessels and increases in blood flow during reactive and functional hyperemia (as in exercise), responses that are adversely affected in certain pathologic conditions. Key words: blood flow velocity, arteriole, endothelium, nitric oxide prostaglandins, reactive oxygen species

The transforming growth factor-beta superfamily member bone morphogenetic protein-2 (BMP-2) is up-regulated in atherosclerotic arteries; however, its effects on the endothelium are not well characterized. Using microdissected coronary arterial endothelial cells (CAECs) and cultured primary CAECs, we demonstrated endothelial mRNA expression of BMP-2 and BMP-4. The proinflammatory cytokine tumor necrosis factor-alpha and H2O2 significantly increased endothelial expression of BMP-2 but not BMP-4. In organ culture, BMP-2 substantially decreased relaxation of rat carotid arteries to acetylcholine and increased production of reactive oxygen species, events inhibited by pharmacologically blocking protein kinase C (PKC) or NAD(P)H oxidase. BMP-2 activated nuclear factor-kappaB in CAECs, and BMP-2 and BMP-4 substantially increased adhesion of monocytic THP-1 cells, which was reduced by pharmacologically inhibiting p42/44 MAP kinase pathway (also by siRNA down-regulating ERK-1/2) or PKC. Incubation of rat carotid arteries with BMP-2 ex vivo also increased adhesion of mononuclear cells to the endothelium, requiring p42/44 MAP kinase and PKC. Western blotting showed that in CAECs and carotid arteries BMP-2 elicited phosphorylation of p42/44 MAP kinase, which was reduced by blocking MAP kinase kinase and PKC. Collectively, expression of BMP-2 is regulated by proinflammatory stimuli, and increased levels of BMP-2 induce endothelial dysfunction, oxidative stress, and endothelial activation. Thus, the proinflammatory effects of BMP-2 may play a role in vascular pathophysiology.

The purpose of the present study was to investigate the effect of NO deficiency on telomerase activity (TA) and telomere length (TL) in mesenteric arteries of 6, 14 and 24-month-old male and female eNOS knockout (KO) and wild type (WT) mice. TA and TL were assessed by the Telo TAGGG Telomerase PCR ELISAPLUS kit and Telo TAGGG Telomere Length Assay kit (Roche). We found that aging continuously and significantly decreased TA. Compared to vessels of 6-month-old mice, TA in vessels of 24-month-old mice decreased by 54 to 75%. There were significant differences in TA between vessels of male and female mice. For example, the optical densities of TA in vessels of 6, 14 and 24-month female WT mice were 0.50, 0.36 and 0.22, whereas in corresponding vessels of male WT mice they were only 0.38, 0.21 and 0.12, respectively. NO deficiency had no significant effect on TA in female vessels, but it significantly decreased TA in male vessels. For example, TA in vessels of 6- and 24-month male KO mice was only 0.31 and 0.08, values which are significantly less than those in corresponding WT vessels. TL in mesenteric arteries was 64 – 80 Kb. There were no significant differences in TL among the groups. We conclude that telomerase activity is mainly influenced by the aging process. Female hormones and/or genetic encoding provide a significant protection of telomerase activity in female vessels. Nitric oxide, derived from eNOS, plays a significant role in the maintenance of telomerase activity in vessels of male mice. (Supported by NIH grants HL-43023, HL-68813 and HL-070653)

We have demonstrated previously that high vascular pressure increases superoxide production in vascular endothelial and smooth muscle layers via a NADPH oxidase-dependent mechanism. In this study, we further investigated the importance of the endothelial cytoskeleton in pressure-induced activation of NADPH oxidase. Mesenteric arteries were isolated from 12-week-old Wistar rats and cannulated in perfusion chambers. Superoxide production was assessed by the SOD-inhibitable, nitro blue tetrazolium (NBT) reduction assay. Vessels were incubated with NBT (100 μM) at 180 mmHg intravascular pressure, with or without inhibitors, for 60 min. We found that intraluminal administration of nocodazole (1 μM, 60min), an agent that selectively disrups endothelial microtubules, decreased pressure-induced superoxide production by 44%. Intraluminal administration of cytochalasin D (1 μM, 60min), an agent that disrupts microfilaments, reduced pressure-induced superoxide production by 46%. Confocal microscopy confirmed that intraluminal administrations of nocodazole or cytochalasin D selectively disassembled the endothelial cytoskeleton and decreased superoxide production in the endothelial layer of the vessels. These results demonstrate that structural integrity of the endothelial cytoskeleton plays a significant role in pressure-induced activation of endothelial NADPH oxidase and the increased production of superoxide. (Supported by NIH grants HL-43023, HL-68813 and HL-070653)

Recent studies suggest that bone morphogenetic protein-2 (BMP-2), a TGFb superfamily member cytokine, is significantly up-regulated in disease-prone vascular beds and atherosclerotic lesions. Despite its pathophysiological importance, the effects of BMP-2 on the endothelium have not been well characterized. We tested the hypothesis that BMP-2 exerts pro-inflammatory effects on endothelial cells. First, we demonstrated mRNA expression of BMP-2 and BMP-4 (a related cytokine) in microdissected coronary arterial endothelial cells and cultured primary coronary arterial endothelial cells (CAECs), which was higher than that in smooth muscle cells. The pro-inflammatory cytokine TNFĄ and H2O2 significantly increased endothelial expression of BMP-2 (but not that of BMP-4). In organ culture, BMP-2 substantially decreased relaxation of rat carotid arteries to acetylcholine and increased production of reactive oxygen species in CAECs and endothelial cells of cultured arteries, which was inhibited by the PKC inhibitor chelerythrine or DPI and apocynin (to block NAD(P)H oxidase). BMP-2 elicited NF-kB activation in CAECs and incubation of carotid arteries with BMP-2 substantially increased adhesion of mononuclear cells to the endothelium. Collectively, expression of BMP-2 is regulated by pro-inflammatory stimuli and increased levels of BMP-2 can induce endothelial dysfunction, oxidative stress and endothelial activation. We propose that the pro-inflammatory endothelial effects of BMP-2 may play a role in vascular pathophysiology. (Grant support: AHA 0430108N, 0435140N, American Federation for Aging Research).

Objective—In response to changes in wall shear stress (WSS) the vascular endothelium releases several factors, among others nitric oxide. On the basis of studies of endothelial cells in culture, suggesting that platelet endothelial cell adhesion molecule-1 (PECAM-1) is specifically involved in sensing and coupling high temporal gradients of fluid shear stress with activation of eNOS, we hypothesized that dilations of isolated skeletal muscle arterioles from PECAM-1 knockout mice (PECAM-KO) will be reduced to rapid increases in WSS elicited by increases in perfusate flow. Methods and Results—Small and large step increases in flow resulted in substantial dilations in arterioles of WT mice (45±4%), but they were markedly reduced in arterioles of PECAM-KO mice (22±5%). The initial slope of dilations, when WSS increased rapidly, was greater in vessels of WT than those of PECAM-KO mice (slopes: 0.378 and 0.094, respectively), whereas the second phase of dilations, when flow/shear stress was steady, was similar in the 2 groups (slopes: 0.085 and 0.094, respectively). Inhibition of eNOS significantly reduced the initial phase of dilations in arterioles from WT, but not from those of PECAM-KO mice. The calcium ionophore A23187 elicited similar NO-mediated dilation in both WT and PECAM-KO mice. Conclusions—In isolated arterioles of PECAM-KO mice activation of eNOS and consequent dilation by agonists is maintained, but the dilation to high temporal gradients of wall shear stress elicited by increases in perfusate flow is reduced. Thus, we propose that PECAM-1 plays an important role in the ability of the endothelium to sense and couple high temporal gradients of wall shear stress to NO-mediated arteriolar dilation during sudden changes in blood flow in vivo.

Background— Hyperhomocysteinemia (HHcy) is a reliable indicator of cardiovascular disease, in part because of the production of superoxide and scavenging of nitric oxide (NO). The present study assessed the impact of HHcy on the NO-dependent control of cardiac O 2 consumption and examined enzymatic sources of superoxide. Methods and Results— Rats and mice were fed methionine in drinking water for 5 to 9 weeks to increase plasma homocysteine, a process that did not cause significant changes in hemodynamic function. The ability of the NO agonists bradykinin and carbachol to reduce myocardial O 2 consumption in vitro was impaired by ≈40% in methionine-fed rats, and this impairment was proportional to their individual plasma homocysteine concentration. However, responses were restored in the presence of ascorbic acid, tempol, and apocynin, which inhibits NADPH oxidase assembly. Western blots showed no difference in Cu/Zn or Mn superoxide dismutase, endothelial NO synthase, or inducible NO synthase protein, but HHcy caused a 100% increase in the p22 phox subunit of NADPH oxidase. Western blots with plasma membrane–enriched fractions of cell lysate detected elevated levels of p22 phox , p67 phox , and rac-1, which indicates increased oxidase assembly. Finally, mice lacking a functional gp91 phox subunit of NADPH oxidase demonstrated normal NO-dependent regulation of myocardial O 2 consumption after methionine feeding. Conclusions— In HHcy, superoxide produced by NADPH oxidase reduces the ability of NO to regulate mitochondrial function in the myocardium. The severity of this effect is proportional to the increase in homocysteine.

Aging impairs shear-stress-dependent dilation of arteries via increased superoxide production, decreased SOD activity, and decreased activation of endothelial nitric oxide (NO) synthase (eNOS). In the present study, we investigated whether chronic increases in shear stress, elicited by increases in blood flow, would improve vascular endothelial function of aged rats. To this end, second-order mesenteric arteries of young (6 mo) and aged (24 mo) male Fischer-344 rats were selectively ligated for 3 wk to elevate blood flow in a first-order artery [high blood flow (HF)]. An in vitro study was then conducted on first-order arteries with HF and normal blood flow (NF) to assess shear stress (1, 10, and 20 dyn/cm2)-induced release of NO into the perfusate. In HF arteries of both age groups, shear stress-induced NO production increased significantly. In 24-mo-old rats, the reduced shear stress-induced NO production in NF arteries was normalized by HF to a level similar to that in NF arteries of 6-mo-old rats. The increased NO production in HF arteries of 24-mo-old rats was associated with increased shear stress-induced dilation, expression of eNOS protein, and shear stress-induced eNOS phosphorylation. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, reduced shear stress-induced eNOS phosphorylation and vasodilation. Superoxide production decreased significantly in HF compared with NF arteries in 24-mo-old rats. The decreased superoxide production was associated with significant increases in CuZn-SOD and extracellular SOD protein expressions and total SOD activity. These results suggest that stimulation with chronic HF restores shear-stress-induced activation of eNOS and antioxidant ability in aged arteries.

A comprehensive study was undertaken to evaluate the effects of inhibition of prostaglandin (PG) synthesis on a variety of reactions in the coronary vascular bed of anesthetized, open-chest dogs. In 23 dogs an electromagnetic flow probe (EMFP) and hydraulic occluder were placed around either the left anterior descending or circumflex branches of the coronary artery and a needle was inserted distal to the EMFP. Injections into the coronary artery of arachidonic acid (AA), bradykinin, adenosine, angiotensin, and PGE2 were given before and after inhibition of PG synthesis by indomethacin (IND) or meclofenamate (MF). The effects of the inhibitors on reactive hyperemia resulting from S-, 10-, 15-, and 20-second occlusions and the dilation resulting from 90-second exposure to 8% O2 were also examined. In each experiment, inhibition of PG synthesis was ascertained by the elimination of vasodilation to AA. After administration of IND or MF, while baseline coronary blood flow was slightly reduced, the total increment of blood flow to vasodilator agents was not significantly altered. Whereas the peak dilation and volume of reactive hyperemia were decreased, the percent flow debt repaid was unchanged and total increment of coronary flow due to hypoxia-induced vasodilation was not significantly modified. Vasoconstrictor responses to angiotensin were also unchanged. These results indicate that while inhibitors of PG synthesis increase coronary resistance, they do not adversely affect vascular responsiveness. We conclude that prostaglandins play little, if any, role in modulating coronary blood flow.

We hypothesized that shear stress stimulates the release of epoxyeicosatrienoic acids (EETs) from arteriolar endothelium, which directly hyperpolarize smooth muscle. To test this hypothesis, a perfusion system, consisting of two separate, serially connected chambers (A and B), was used. A donor vessel, isolated from gracilis muscle of female NO-deficient mice and rats, was cannulated in chamber A. In chamber B, an endothelium-denuded detector vessel isolated from mesentery of these animals was cannulated. In the presence of indomethacin, 5, 10, and 20 dyne/cm 2 shear stress elicited dilation of donor vessels, followed by dilation of detector vessels. Changes in membrane potential of the detector vessel smooth muscle cells in response to the perfusate from 5 and 10 dyne/cm 2 shear stress–stimulated donor vessels was also recorded (by ≈−12 to −15 and −20 to −30 mV, respectively). Exposing detector vessels to 30 mmol/L KCl or pretreating them with iberiotoxin abolished their hyperpolarization and dilation to the flow of perfusate. Pretreatment of donor vessels with PPOH, an inhibitor of cytochrome P-450/epoxygenase, eliminated dilator responses in both donor and detector vessels, as well as the hyperpolarization of detector vessels. GC-MS analysis showed increasing release of EETs into the perfusate collected from 1, 5, and 10 dyne/cm 2 shear stress–stimulated arterioles, which was abolished by PPOH. Thus, EETs, released from endothelial cells of donor vessels stimulated with shear stress, hyperpolarize smooth muscle of downstream detector vessels, confirming their identity as endothelium-derived hyperpolarizing factors and suggesting that gap junctional communication may not be necessary for shear stress–stimulated EDHF-mediated vasodilation.

Background— We have reported that there is a limitation of exercise capacity in mice with defects in the expression of endothelial nitric oxide (NO) synthase, which is associated with a greater increase in whole-body oxygen consumption (V̇ o 2 ). We hypothesized that in states in which superoxide anion (O 2 − ) is increased, especially in the mitochondria, whole-body V̇ o 2 will be increased because of the inactivation of NO, and consequently, exercise capacity will be reduced. Methods and Results— Heterozygous manganese superoxide anion dismutase (SOD2) gene–knockout mice (SOD2 +/− ), in which SOD2 activity is reduced by 30% to 80%, and wild-type control mice (SOD2 +/+ ) were treadmill-tested to measure indices defining exercise capacity. Tempol was given to each mouse for 7 days by an intraperitoneal injection to scavenge O 2 − before a second treadmill testing. V̇ o 2 and carbon dioxide production (V̇ co 2 ) at rest were increased in SOD2 +/− . The work (vertical distance run × body weight) to exhaustion was decreased in SOD2 +/− . When the maximum V̇ o 2 and V̇ co 2 were corrected to per work unit, they were increased in SOD2 +/− . Tempol normalized basal V̇ o 2 and V̇ co 2 and improved the work to exhaustion and corrected V̇ o 2 and V̇ co 2 in SOD2 +/− . V̇ o 2 of skeletal muscle was measured in vitro. Bradykinin-induced reduction in V̇ o 2 in vitro was attenuated in SOD2 +/− , and was acutely restored by Tempol. There was a decrease in SOD2 protein level and a concomitant increase in lucigenin-detectable O 2 − production in skeletal muscle from SOD2 +/− . Conclusions— These results suggest that exercise capacity is reduced in conditions in which superoxide anion is increased, and this is associated with a greater increase in whole-body oxygen consumption in SOD2 +/− compared with SOD2 +/+ .

Background—Recent studies suggest that bone morphogenetic protein-2 (BMP-2), a transforming growth factor-&bgr; superfamily member cytokine, plays an important role both in vascular development and pathophysiological processes, including endothelial activation that is likely to contribute to the development of coronary atherosclerosis, yet the factors that regulate arterial expression of BMP-2 are completely unknown. We tested the hypothesis that BMP-2 expression in endothelial cells is governed by an H2O2 and nuclear factor (NF)-&kgr;&Bgr;–dependent pathway that can be activated by both proinflammatory and mechanical stimuli. Methods and Results—The proinflammatory cytokine tumor necrosis factor (TNF)-&agr; induced NF-&kgr;&Bgr; activation and elicited significant increases in BMP-2 mRNA and protein in primary coronary arterial endothelial cells and human umbilical vein endothelial cells that were prevented by NF-&kgr;&Bgr; inhibitors (pyrrolidine dithiocarbamate and SN-50), silencing of p65 (siRNA), or catalase. Administration of H2O2 also elicited NF-&kgr;&Bgr; activation and BMP-2 induction. In organ culture, exposure of rat arteries to high pressure (160 mm Hg) elicited H2O2 production, nuclear translocation of NF-&kgr;&Bgr;, and upregulation of BMP-2 expression. Although high pressure upregulated TNF-&agr;, it appears that it directly regulates BMP-2 expression, because upregulation of BMP-2 was also observed in vessels of TNF-&agr; knockout mice. Conclusions—Vascular BMP-2 expression can be regulated by H2O2-mediated activation of NF-&kgr;&Bgr; both by inflammatory stimuli and by high intravascular pressure.