Vascular aging: Chronic oxidative stress and impairment of redox signaling—consequences for vascular homeostasis and disease

Figures & data

Figure 1. General aspects of RNOS in aging. Young individuals have a higher stress tolerance due to a high antioxidant potential, and efficient repair mechanisms restore homeostatic conditions in response to damage. Under these physiological conditions redox regulation can normally proceed in special compartments but differs in a cell type-specific manner. Prostaglandin endoperoxide H₂ synthase (PGHS, COX) is an excellent example of a highly redox-controlled enzyme. In aging the stress tolerance to RNOS decreases. The molecular basis for the loss of balance between damage and repair is the enhanced RNOS generation which must be compensated by antioxidant systems. These exhibit a reduced efficiency in repair with increasing age. As a consequence, oxidatively altered macromolecules accumulate and burden physiological cellular processes. This attenuates redox regulation and also interferes with intercellular communication.

Table I. Theories of aging.

Theory	Definition
Evolutionary concept of antagonistic pleiotrophy	Natural selection favors genes that act early in life and increase reproduction, rather than genes that act to preserve non-germ cells (the ‘disposable soma’). Principally a function of external factors.
Somatic mutation theory of aging	Accumulation of DNA mutations during life is responsible for degenerative senescence caused by loss of tumor suppressor gene function and by point mutations in pro-oncogenes.
	• Not valid for postmitotic tissues like heart and neurons
	• ROS are putative mutagens
	• DNA repair capacity and efficacy correlate with the species-specific lifespan
Replicative or intrinsic senescence introduced by Hayflick and Moorhead (1961) or Telomere hypothesis McClintock and Müller; discovery of telomeres (1930s)	Cells react on subculture with a cessation of cell division, the ‘Hayflick limit’ (24,35,44,45).
	• Morphological parameters: increase in cell size, polymorphic nuclei with transcriptionally inactive heterochromatic structures (senescence-associated heterochromatic foci = SAHF), cell flattening (‘fried egg appearance’, cell enlargement, and vacuolization) and increase in lysosomal mass
	• Molecular parameters: attrition of telomeres and genomic instability (‘crisis’) due to cross-linkage of chromosomes, induction, stabilization, and activation of cell cycle activators (p21, p16INK4a, p53, pRB, p27, p15, p19ARF), persistent Ras activation and MAP-kinase cascade stimulation and expression of senescence-associated β-galactosidase (36–38)
	• Adult cells have limited capacity to prevent telomere shortening (telomerase or alternative lengthening of telomeres (ALT)) resulting in telomere shortening with age and growth arrest (25–27)
Free radical theory of aging introduced by Gerschman et al. (1954) and Harman (1956)	Lifelong primarily mitochondria-derived oxidative stress negatively affects biological macromolecules leading to their accumulation, modification, and depletion and to an acceleration of the oxidant flux (55,56,244).
	• ˜0.1% of the utilized oxygen is converted into ROS
	• Accumulation of oxidatively modified molecules inversely correlates with the lifespan of the species
	• Impaired or dysfunctional lysosomes and proteasomes
	• Modifications include advanced glycated end-products (AGE), lipofuscin (62,65), protein carbonyls (61), dityrosine, nitrotyrosine, protein cross-linking of extracellular matrix components causing matrix stiffness, oxidatively modified DNA bases such as 8-oxo-d-guanosine (245) or 8-nitro-d-guanosine, cross-linked protein-lipid products (HNE-protein), DOPA-protein cross-links, modified sulfhydryls (glutathiolated, sulfonic acid, methionine sulfoxide, etc.)
Mitochondrial theory of aging introduced by Miquel (1980s) and Harman	Organelle dysfunction increases with age and has profound negative pleiotropic effects on cell physiology (71).
	• Energy depletion, Ca^2 + dysregulation, increased apoptosis rate, increased ROS formation
	• The close proximity of ROS formation to mitochondrial DNA leads to faster accumulation of mutations and deletions in postmitotic cells causing mitochondrial dysfunction (87,88,98,99,100)
Rate of living—caloric restriction	Species with a higher metabolic rate have a shorter maximum lifespan potential (MLSP) (228)
	• Higher energy consumption leads to higher O2 utilization in cell respiration and to a higher rate of mitochondrial free radical generation
	• Caloric restriction is a model which leads to lifespan prolongation. Molecular mechanisms may involve the activation of sirtuins or AMP kinase (sensors of energy status), which can be pharmacologically manipulated by polyphenol treatment including resveratrol

Table II. Major cellular NO sources and regulation.

Source	Description	Capacity
For all NO synthases	• Co-factors: FAD, FMN, BH4, Heme, P450 enzyme, homo dimer, Zn-finger, Ca^2 + /calmodulin
	• L-Arg + NADPH + H ^+ + O2 → NOHLA + NADP ^+ + H2O
	• NOHLA + ½ NADPH + ½ H ^+ + O2 → L-citrulline + ½ NADP ^+ + ^•NO + H2O (115)
NOS-1/nNOS/neuronal NO synthase (160 kDa)	• Regulation via Ca^2 + /calmodulin (119)	High
	• Splice variants—mitochondrial NO synthase
	• Mainly nervous system and splice variants in skeletal and vascular smooth muscle (nNOSμ) (246,247)
	• N-terminal PDZ-domain that is important for interaction with syntrophin. Attachment of nNOS to membranes (plasma, sarcolemma, endoplasmatic reticulum) via dystoglycan, dystrophin, and dystrobrevin complexes or in neurons association with NMDA receptor via PSD-95, Capon and Dexras
	• Caveolin III, phosphofructokinase M
NOS-2/iNOS/inducible NO synthase (125–135 kDa)	• Ca^2 + /calmodulin is tightly bound to the enzyme and leads to constitutive activation	High
	• Cytosolic
	• Induced as early immediate gene on inflammatory stimuli such as LPS or cytokines
NOS-3/eNOS/endothelial NO synthase (135 kDa)	• Vascular endothelium (117)	Low
	• Regulation via Ca^2 + /calmodulin (10–20-fold change in activity)
	• Caveolin I association inhibits the enzyme—‘caveolar paradox’
	• Phosphorylation at Ser1177 (positive regulator at the reductase domain) and Thr495 (negative regulator at the Ca^2 + /calmodulin binding domain) can cause activity changes up to 2–3-fold. Function: Vascular adaptation to shear stress or reaction on stimuli such as estrogen, insulin, bradykinin, or vascular endothelial growth factor (VEGF) (248)
	• Phosphorylation by Akt/PKB and PKA, PKC
	• Palmitoylation and myristoylation target the enzyme to the plasma membrane
	• Association with HSP90, bradykinin receptor, CAT-2
Mitochondria	• Putative mitochondrial NO synthases (NOS-3 (heart) or a NOS-1 splice variant)	Low
	• Reduction of nitrite at complex III—under hypoxia (249)
Xanthine oxidase/aldehyde dehydrogenase	• Reduction of nitrite to ^•NO under hypoxic conditions—putative autoregulatory mechanism (250,251)	Low
Hemoglobin/myoglobin	• Reduction of nitrite to ^•NO under hypoxic conditions—putative autoregulatory mechanism for capillaries and muscle (252,253)	Medium

Figure 2. The vascular ^•NO signaling cascade and the antagonizing ^•O₂⁻. Agonist stimulation of endothelial cells, including angiotensin II, acetylcholine, serotonin, etc., or shear stress causes activation of endothelial NO synthase by a transient increase in intracellular calcium or phosphorylation. ^•NO as a freely diffusible molecule can easily cross cell membranes and activate soluble guanylyl cyclase in smooth muscle cells, increasing intracellular cGMP levels. Protein kinase G is activated and can cause a decrease in intracellular calcium [Ca^{2 +} ]_i and mediates the dephosphorylation of myosin light chain leading to vasorelaxation. Superoxide can react with and reduce free ^•NO, impairing the ^•NO-cGMP signaling pathway. Inactivation of soluble guanylyl cyclase by superoxide has been reported, too. Various superoxide sources in the endothelium and vascular smooth muscle cells have been identified, including mitochondria, uncoupled NO synthase, xanthine oxidase, NADPH oxidases, and epoxygenase. Depending on the model and pathology, different sources of ^•O₂⁻ can be activated. (Ag = agonist; [Ca^{2 +} ]_i = intracellular calcium; NOS-3 = endothelial NO synthase; sGC = soluble guanylyl cyclase; ^•NO = nitric oxide; cGMP = cyclic guanosine monophosphate; Rec = receptor; ^•O₂⁻ = superoxide anion)

Table III. Potential sources of superoxide in the vasculature.

Source	Description	Capacity
Uncoupled eNOS	Confirmed: Uncoupling occurs under reduced availability of either L-arginine or tetrahydrobiopterin (BH4) (133,207).	Low
	Unconfirmed:
	• Low temperature SDS–PAGE monomer and dimer distribution as an indicator of the status of the Zn-finger, involved in uncoupling (134)
	• Phosphorylation of Ser1177 (254)
	• Heat shock protein 90 (HSP90) and its inhibitor geldanamycin. Recent data indicate that HSP90 can improve NO release but does not contribute to eNOS uncoupling (255)
NADPH oxidases (NOX = homologs of gp91^phox)	Important isoforms in the vasculature (256–258):	In the vascular system low, but in neutrophiles high (oxidative burst)
	• NOX-1 = MOX 1 participates in the regulation of cell proliferation via activation of MAP-kinase cascade. Separate cytosolic components
	• NOX-2 = gp91^phox, homologous to the phagocyte oxidase but with lower ^•O2^− and intracellular output. Forms upon activation with a complex with membrane (gp22^phox) and cytosolic (p40^phox, p47^phox, p67^phox, and Rac 1, 2) components
	• NOX-4
	Induction and activation via angiotensin II.
	Inhibition of activation via inhibitors which interfere with the complex formation such as apocynin, gliotoxin, or phenylarsene oxide (PAO)
	Positive ^•O2^− feedback via PKC.
Xanthine oxidase	Conversion of the xanthine dehydrogenase into an oxidase by (259):	Medium, depending on its substrate availability
	• Reversible and irreversible oxidation of essential sulfhydryls
	• Proteolysis
	Extracellular xanthine oxidase is synthesized in the liver and sticks via proteoglycans to the endothelial surface.
Mitochondria	Superoxide sources (73,74,144–147):	High, up to 0.1% of utilized O2
	• Complex I ubiquinone binding site
	• Complex III—accumulation of ubisemiquinone
	To date it is unclear if mitochondrial ^•O2^− generation is accidental or can be regulated by e.g. mitochondrial anion channel, uncoupling proteins (UCP), oxidative modifications of complex I, or inhibition of complex IV via ^•NO.

Figure 3. The Yin–Yang system of prostanoids. Agonist stimulation of endothelial cells causes an increase in intracellular calcium levels [Ca^{2 +} ]_i, which releases arachidonic acid via activation of phospholipase A₂ from membrane phospholipids. Alternatively, phospholipase A₂ is regulated by fluid or cyclic shear stress through phosphorylation. Arachidonic acid is converted by prostaglandin endoperoxide H₂ synthase (cyclo-oxygenase) into the intermediate prostaglandin endoperoxide H₂, which is further processed into prostacyclin by prostacyclin synthase. Prostacyclin mediates vasorelaxation by acting on the prostacyclin receptor, activating adenylyl cyclase, and elevating cyclic AMP, resulting in an increase in protein kinase A activity. Furthermore, prostacyclin inhibits platelet aggregation and leukocyte adhesion to the endothelium. Under conditions that cause inhibition of prostacyclin synthase, PGH₂ levels increase and PGH₂ exerts thromboxane-like effects via stimulation of the thromboxane A₂/PGH₂ receptor. Thromboxane A₂ stimulates platelet aggregation and enhances this process in an autocrine loop or promotes leukocyte adhesion. Activation of the thromboxane A₂/PGH₂ receptor leads to activation of phospholipase C and an increase in intracellular calcium levels. (AA = arachidonic acid; AC = adenylyl cyclase; Ag = agonist; [Ca^{2 +} ]_i = intracellular calcium; G = G-protein; ONOO⁻ = peroxynitrite; ^•O₂⁻ = superoxide; PLA₂ = phospholipase A₂; ^•NO = nitric oxide; PGHS-1 = prostaglandin endoperoxide H₂ synthase; PGH₂ = prostaglandin endoperoxide H₂; PGIS = prostacyclin synthase; PLC = phospholipase C; PKA = protein kinase A; Rec = receptor; TxA₂ = thromboxane A₂; TxAS = thromboxane synthase)

Figure 4. Free radical-mediated platelet hyperreactivity in aging. Platelets use peroxynitrite at low concentrations (< 21 nM) in order to provide the peroxide tone for modulating basal prostaglandin endoperoxide H₂ synthase (cyclo-oxygenase-1) activity after agonist-mediated platelet activation. NADPH oxidase and endothelial NO synthase concertedly release superoxide anion and ^•NO to form peroxynitrite. Cyclo-oxygenase-1 provides the substrate prostaglandin endoperoxide H₂ for thromboxane synthase, which is converted into thromboxane A₂. This potent eicosanoid further activates platelets in an autocrine loop or causes the recruitment of other platelets to the thrombus. However, aging leads to an elevated superoxide formation via uncoupling of NO synthase, conversion of xanthine dehydrogenase into an oxidase, activation of NADPH oxidase, or by electron leakage in the mitochondrial electron transport chain. This saturates the platelet peroxide tone, resulting in loss of the modulation of COX-1 activity, which subsequently leads to hyperreactivity of platelets. Therefore, platelet aggregation readily occurs at lower doses of agonists and proceeds faster. (Ag = agonist; Rec = receptor; PLA₂ = phospholipase A₂; PGHS-1 = prostaglandin endoperoxide H₂ synthase; AA = arachidonic acid; PGH₂ = prostaglandin endoperoxide H₂; TxAS = thromboxane synthase; ONOO⁻ = peroxynitrite; TxA₂ = thromboxane A₂; [Ca^{2 +} ]_i = intracellular calcium; ^•NO = nitric oxide; ^•O₂⁻ = superoxide)

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