Effect of oxidative stress on telomere maintenance in aortic smooth muscle cells

Reactive oxygen species (ROS) and telomere dysfunction are both associated with aging and the development of age-related diseases. Although there is evidence for a direct relationship between ROS and telomere dysfunction as well as an independent association of oxidative stress and telomere attrition with age-related disorders, there has not been sufficient exploration of how the interaction between oxidative stress and telomere function may contribute to the pathophysiology of cardiovascular diseases (CVD). To better understand the complex relationships between oxidative stress, telomerase biology and pathophysiology, we examined the telomere biology of aortic smooth muscle cells (ASMCs) isolated from mutant mouse models of oxidative stress. We discovered that telomere lengths were significantly shorter in ASMCs isolated from superoxide dismutase 2 heterozygous (Sod2^+/?) mice, which exhibit increased arterial stiffness with aging, and the observed telomere attrition occurred over time. Furthermore, the telomere erosion occurred even though telomerase activity increased. In contrast, telomeres remained stable in wild-type and superoxide dismutase 1 heterozygous (Sod1^+/?) mice, which do not exhibit CVD phenotypes. The data indicate that mitochondrial oxidative stress, in particular elevated superoxide levels and decreased hydrogen peroxide levels, induces telomere erosion in the ASMCs of the Sod2^+/? mice. This reduction in telomere length occurs despite an increase in telomerase activity and correlates with the onset of disease phenotype. Our results suggest that the oxidative stress caused by imbalance in mitochondrial ROS, from deficient SOD2 activity as a model for mitochondrial dysfunction results in telomere dysfunction, which may contribute to pathogenesis of CVD.     

Endothelial and smooth muscle cells from abdominal aortic aneurysm have increased oxidative stress and telomere attrition

Background

Abdominal aortic aneurysm (AAA) is a complex multi-factorial disease with life-threatening complications. AAA is typically asymptomatic and its rupture is associated with high mortality rate. Both environmental and genetic risk factors are involved in AAA pathogenesis. Aim of this study was to investigate telomere length (TL) and oxidative DNA damage in paired blood lymphocytes, aortic endothelial cells (EC), vascular smooth muscle cells (VSMC), and epidermal cells from patients with AAA in comparison with matched controls.

Methods

TL was assessed using a modification of quantitative (Q)-FISH in combination with immunofluorescence for CD31 or α-smooth muscle actin to detect EC and VSMC, respectively. Oxidative DNA damage was investigated by immunofluorescence staining for 7, 8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG).

Results and Conclusions

Telomeres were found to be significantly shortened in EC, VSMC, keratinocytes and blood lymphocytes from AAA patients compared to matched controls. 8-oxo-dG immunoreactivity, indicative of oxidative DNA damage, was detected at higher levels in all of the above cell types from AAA patients compared to matched controls. Increased DNA double strand breaks were detected in AAA patients vs controls by nuclear staining for γ-H2AX histone. There was statistically significant inverse correlation between TL and accumulation of oxidative DNA damage in blood lymphocytes from AAA patients. This study shows for the first time that EC and VSMC from AAA have shortened telomeres and oxidative DNA damage. Similar findings were obtained with circulating lymphocytes and keratinocytes, indicating the systemic nature of the disease. Potential translational implications of these findings are discussed.     

Vascular smooth muscle cells direct extracellular dysregulation in aortic stiffening of hypertensive rats

Aortic stiffening is an independent risk factor that underlies cardiovascular morbidity in the elderly. We have previously shown that intrinsic mechanical properties of vascular smooth muscle cells (VSMCs) play a key role in aortic stiffening in both aging and hypertension. Here, we test the hypothesis that VSMCs also contribute to aortic stiffening through their extracellular effects. Aortic stiffening was confirmed in spontaneously hypertensive rats (SHRs) vs. Wistar‐Kyoto (WKY) rats in vivo by echocardiography and ex vivo by isometric force measurements in isolated de‐endothelized aortic vessel segments. Vascular smooth muscle cells were isolated from thoracic aorta and embedded in a collagen I matrix in an in vitro 3D model to form reconstituted vessels. Reconstituted vessel segments made with SHR VSMCs were significantly stiffer than vessels made with WKY VSMCs. SHR VSMCs in the reconstituted vessels exhibited different morphologies and diminished adaptability to stretch compared to WKY VSMCs, implying dual effects on both static and dynamic stiffness. SHR VSMCs increased the synthesis of collagen and induced collagen fibril disorganization in reconstituted vessels. Mechanistically, compared to WKY VSMCs, SHR VSMCs exhibited an increase in the levels of active integrin β1‐ and bone morphogenetic protein 1 (BMP1)‐mediated proteolytic cleavage of lysyl oxidase (LOX). These VSMC‐induced alterations in the SHR were attenuated by an inhibitor of serum response factor (SRF)/myocardin. Therefore, SHR VSMCs exhibit extracellular dysregulation through modulating integrin β1 and BMP1/LOX via SRF/myocardin signaling in aortic stiffening.     

Nitrotyrosine promotes human aortic smooth muscle cell migration through oxidative stress and ERK1/2 activation

Nitrotyrosine is a new biomarker of atherosclerosis and inflammation. The objective of this study was to determine the direct effects of free nitrotyrosine on human aortic smooth muscle cell (AoSMC) migration and molecular mechanisms. By a modified Boyden chamber assay, nitrotyrosine significantly increased AoSMC migration in a concentration-dependent manner. For example, nitrotyrosine at 300 nM increased AoSMC migration up to 152% compared with l-tyrosine-treated control cells (P<0.01). Cell wound healing assay confirmed this effect. Nitrotyrosine significantly increased the expression of some key cell migration-related molecules including PDGF receptor-B, matrix metalloproteinase 2 (MMP2) and integrins alphaV and beta3 at both mRNA and protein levels in AoSMC (P<0.01). In addition, nitrotyrosine increased reactive oxygen species (ROS) production in AoSMC by staining with fluorescent dye DCFHDA. Furthermore, nitrotyrosine induced transient phosphorylation of ERK2 by Bio-Plex luminex immunoassay and western blot analysis. AoSMC were able to uptake nitrotyrosine. Antioxidants including seleno-l-methionine and superoxide dismutase mimetic (MnTBAP) as well as ERK1/2 inhibitor PD98059 effectively blocked the promoting effect of nitrotyrosine on AoSMC migration and the mRNA expression of above cell migration-related molecules. Thus, nitrotyrosine directly increases AoSMC migration in vitro and the expression of migration-related molecules through overproduction of ROS and activation of ERK1/2 pathway. Nitrotyrosine may contribute to cardiovascular pathogenesis.     

Reduced levels of cyclic AMP contribute to the enhanced oxidative stress in vascular smooth muscle cells from spontaneously hypertensive rats

We have earlier shown that aortic vascular smooth muscle cells (VSMC) from 12-week-old spontaneously hypertensive rats (SHR) exhibited enhanced production of superoxide anion (O(2)(-)) compared with Wistar-Kyoto (WKY) rats. This production was attenuated to control levels by losartan, an angiotensin II (Ang II) AT(1)-receptor antagonist, suggesting that the AT(1) receptor is implicated in enhanced oxidative stress in SHR. Since AT(1) receptor activation signals via adenylyl cyclase inhibition and decreases cAMP levels, it is possible that AT(1) receptor-mediated decreased levels of cAMP contribute to the enhanced production of O(2)(-) in SHR. The present study was undertaken to investigate this possibility. The basal adenylyl cyclase activity as well as isoproterenol and forskolin-mediated stimulation of adenylyl cyclase was significantly attenuated in VSMC from 12-week-old SHR compared with those from WKY rats, whereas Ang II-mediated inhibition of adenylyl cyclase was significantly enhanced by about 70%, resulting in decreased levels of cAMP in SHR. NADPH oxidase activity and the levels of O2- were significantly higher (about 120% and 200%, respectively) in VSMC from SHR than from WKY rats. In addition, the levels of p47(phox) and Nox4 proteins, subunits of NADPH oxidase, were significantly augmented about 35%-40% in VSMC from SHR compared with those from WKY rats. Treatment of VSMC from SHR with 8Br-cAMP, as well as with cAMP-elevating agents such as isoproterenol and forskolin, restored to control WKY levels the enhanced activity of NADPH oxidase and the enhanced levels of O(2)(-), p47(phox), and Nox4. Furthermore, in the VSMC A10 cell line, 8Br-cAMP also restored the Ang II-evoked enhanced production of O(2)(-), NADPH oxidase activity, and enhanced levels of p47(phox) and Nox4 proteins to control levels. These data suggest that decreased levels of cAMP in SHR may contribute to the enhanced oxidative stress in SHR and that increasing the levels of cAMP may have a protective effect in reducing oxidative stress and thereby improve vascular function.     

Decreased sensitivity of spontaneously hypertensive rat aortic smooth muscle to vasorelaxation by atriopeptin III

The effects of a synthetic form of Atrial Natriuretic Factor (ANF) on spontaneously hypertensive rat aortic smooth muscle were investigated using either an alpha-adrenoceptive agonist (phenylephrine) or an agent which partially depolarized the plasma membrane (20mM KCl) as a contractile agent. The relaxant response was studied under conditions resembling normal physiological calcium ion levels (1.5mM) as well as over a range of calcium ion concentrations (0.1-2.5mM). The results demonstrate a hyporesponsiveness of hypertensive aorta to vasorelaxation induced by synthetic ANF, which is more apparent when the tissue is contracted with KCl. The results also suggest that ANF, which has been shown previously to inhibit intracellular and receptor operated calcium channel mobilization only, may additionally work through a mechanism which is related to the voltage induced calcium flux across the membrane, which also is inhibited less in hypertensive smooth muscle.     

Force-velocity relationships in hypertensive arterial smooth muscle

Increased total peripheral resistance is the cardinal haemodynamic disorder in essential hypertension. This could be secondary to alterations in the mechanical properties of vascular smooth muscle. Adequate study has not been made of the force-velocity (F-V) relationship in hypertensive arterial smooth muscle. Increased shortening in arterial smooth muscle would result in greater narrowing of arteries. The objectives of this investigation were to see if there is (i) increased shortening or increased maximum change in muscle length (delta Lmax where L stands for muscle length), (ii) an increased maximum velocity of shortening (Vmax) measured in l omicron per second where l omicron is the optimal muscle length for tension development, and (iii) a difference in maximum isometric tension (P omicron) developed in spontaneously hypertensive rat (SHR; N = 6) compared with normotensive Wistar Kyoto rat (WKY;N = 5) caudal artery strips. An electromagnetic muscle lever was employed in recording force-velocity data. Analysis of these data revealed the following: (a) the SHR mean P omicron of 6.21 +/- 1.01 N/cm2 was not different from the mean WKY P omicron of 6.97 +/- 1.64 N/cm2 (p greater than 0.05); (b) the SHR preparations showed greater shortening for all loads imposed; (c) the SHR Vmax of 0.016 l omicron/s was greater than the WKY Vmax of 0.013 l omicron/s (p less than 0.05). This study provides evidence that while hypertensive arterial smooth muscle is not able to produce more force than normotensive arterial smooth muscle, it is capable of faster and greater shortening. The latter could result in increased narrowing of hypertensive arteries and increased blood pressure.     

Identification of oxidative stress-regulated genes in rat aortic smooth muscle cells by suppression subtractive hybridization

Transfer RNA modification improves the rate of aa-tRNA selection at the A-site and the fitness in the P-site and thereby prevents frameshifting according to a new model how frameshifting occurs [Qian et al. (1998) Mol. Cell 1, 471-482]. Evidence that the presence of various modified nucleosides in tRNA also influences central metabolism, thiamine metabolism, and bacterial virulence is reviewed.     

Pure pressure stress increased monocarboxylate transporter in human aortic smooth muscle cell membrane

Lactate is formed and utilized continuously under fully aerobic conditions. Lactate is oxidized actively at all times, especially during exercise. Family of monocarboxylate transport proteins (MCTs) that are differentially expressed in cells and tissues accomplishes the facilitated transport of lactate across membranes. Previously we reported that there is MCT1 in blood circulation. We also reported the pressure stress stimulated cell proliferation in aortic smooth muscle cells (HASMC). In this experiment we attempted to prove the existence of MCT1 in HASMC and to clarify the effect of pressure stress on MCT1 localization in HASMC. We determined succinate dehydrogenase (SDH) activity as a marker of energy metabolism in cells. SDH activity was increased by pressure stress. Lactate enhanced the SDH activity under pressure stress (160 mmHg for 3 h) as dose dependent manner. On the other hand, lactate excretion was suppressed by the addition of lactate. We could detect MCT1 in the cytosolic and the membrane fractions of HASMC. The pressure stress increased MCT1 in the membrane fraction in the presence of extracellular lactate. In summary, we proved the existence of MCT1 in HASMC. Pressure stress changed the localization of MCT1. The increased membranous MCT1 may transport lactate for energy metabolism in cells.     

Proteomic profiling and identification of cofilin responding to oxidative stress in vascular smooth muscle

We used 2-DE and MALDI-TOF/TOF to identify proteins of vascular smooth muscle cells whose expression was or was not altered by exposure to 500 microM H2O2 for 30 min. We detected more than 800 proteins on silver-stained gels of whole protein extracts from rat aortic smooth muscle strips. Of these proteins, 135 clearly unaffected and 19 having levels altered by exposure to H2O2 were identified. Protein characterization revealed that the most prominent vascular smooth muscle proteins were those with antioxidant, cytoskeletal structure, or muscle contraction. In addition, cofilin, an isoform of the actin depolymerizing factor family, shifted to its basic site on the 2-DE gel as a result of H2O2 treatment. In Western blot analysis of proteins from A7r5 aortic smooth muscle cells, the phosphorylation, but not the expression, of cofilin was decreased by H2O2 in a dose-dependent manner. The H2O2-induced dephosphorylation of cofilin and apoptosis was inhibited by Na3VO4, an inhibitor of protein tyrosine phosphatase (PTP). These results suggest that cofilin is one of the proteins regulated by H2O2 treatment in vascular smooth muscle, and has an important role in the induction of vascular apoptosis through PTP-dependent mechanisms.     

Aging, oxidative responses, and proliferative capacity in cultured mouse aortic smooth muscle cells

The cellular mechanisms that contribute to the acceleration of atherosclerosis in aging populations are poorly understood, although it is hypothesized that changes in the proliferative capacity of vascular smooth muscle cells is contributory. We addressed the relationship among aging, generation of reactive oxygen species (ROS), and proliferation in primary culture smooth muscle cells (SMC) derived from the aortas of young (4 mo old) and aged (16 mo old) mice to understand the phenotypic modulation of these cells as aging occurs. SMC from aged mice had decreased proliferative capacity in response to alpha-thrombin stimulation, yet generated higher levels of ROS and had constitutively increased mitogen-activated protein kinase activity, in comparison with cells from younger mice. These effects may be explained by dysregulation of cell cycle-associated proteins such as cyclin D1 and p27Kip1 in SMC from aged mice. Increased ROS generation was associated with decreased endogenous antioxidant activity, increased lipid peroxidation, and mitochondrial DNA damage. Accrual of oxidant-induced damage and decreased proliferative capacity in SMC may explain, in part, the age-associated transition to plaque instability in humans with atherosclerosis.     

L-carnitine attenuates oxidative stress in hypertensive rats

The present study aimed to investigate whether l-carnitine (LC) protects the vascular endothelium and tissues against oxidative damage in hypertension. Antioxidant enzyme activities, glutathione and lipid peroxidation were measured in the liver and heart of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. Nitrite and nitrate levels and total antioxidant status (TAS) were evaluated in plasma, and the expression of endothelial nitric oxide synthase (eNOS) and p22phox subunit of NAD(P)H oxidase was determined in aorta. Glutathione peroxidase activity was lower in SHR than in WKY rats, and LC increased this activity in SHR up to values close to those observed in normotensive animals. Glutathione reductase and catalase activities, which were higher in SHR, tended to increase after LC treatment. No differences were found in the activity of superoxide dismutase among any animal group. The ratio between reduced and oxidized glutathione and the levels of lipid peroxidation were respectively decreased and increased in hypertensive rats, and both parameters were normalized after the treatment. Similarly, LC was able to reverse the reduced plasma nitrite and nitrate levels and TAS observed in SHR. We found no alterations in the expression of aortic eNOS among any group; however, p22phox mRNA levels showed an increase in SHR that was reversed by LC. In conclusion, chronic administration of LC leads to an increase in hepatic and cardiac antioxidant defense and a reduction in the systemic oxidative process in SHR. Therefore, LC might increase NO availability in SHR aorta by a reduction in superoxide anion production.     

Proliferation kinetics of aortic smooth muscle cell populations: comparison of normotensive and spontaneously hypertensive rats

Abstract. An in vitro autoradiographic study of the proliferation of smooth muscle cells (SMC) from the aorta of normotensive and spontaneously hypertensive rats has been made. It was found, in primary culture, that SMC of spontaneously hypertensive rats entered proliferation at 2-2.5 days later than those from normotensive animals. As revealed by their very intensive labelling, a subpopulation of SMC with a high turnover rate was found in primary culture. In freshly isolated SMC from normotensive rat aorta, a subpopulation in S phase was detected, but we failed to detect it in aortae from spontaneously hypertensive rats. A difference in proliferative behaviour was also observed in subcultures of SMC from rats of both strains.     

Proliferation kinetics of aortic smooth muscle cell populations: comparison of normotensive and spontaneously hypertensive rats

An in vitro autoradiographic study of the proliferation of smooth muscle cells (SMC) from the aorta of normotensive and spontaneously hypertensive rats has been made. It was found, in primary culture, that SMC of spontaneously hypertensive rats entered proliferation at 2-2.5 days later than those from normotensive animals. As revealed by their very intensive labelling, a subpopulation of SMC with a high turnover rate was found in primary culture. In freshly isolated SMC from normotensive rat aorta, a subpopulation in S phase was detected, but we failed to detect it in aortae from spontaneously hypertensive rats. A difference in proliferative behaviour was also observed in subcultures of SMC from rats of both strains.