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[Engadin]

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S O U R C E :   Science Direct

 

 

 

 

Highlights

 

 

•  Aortic stiffness in aging is associated with increased mitochondrial NOX4 levels.

 

•  Young mitochondrial Nox4 overexpressing transgenic mice phenocopy aortic stiffness.

 

•  Nox4 transgenic mice show increased VSMC stiffness, aortic remodeling and fibrosis.

 

•  Nox4 transgenic mouse VSMC show DNA damage, senescence, apoptosis and inflammation.

 

•  High mitochondrial catalase levels blunt aortic stiffness in Nox4 transgenic mice.

 

 

 

Abstract

 

Aging is characterized by increased aortic stiffness, an early, independent predictor and cause of cardiovascular disease. Oxidative stress from excess reactive oxygen species (ROS) production increases with age. Mitochondria and NADPH oxidases (NOXs) are two major sources of ROS in cardiovascular system. We showed previously that increased mitochondrial ROS levels over a lifetime induce aortic stiffening in a mouse oxidative stress model. Also, NADPH oxidase 4 (NOX4) expression and ROS levels increase with age in aortas, aortic vascular smooth muscle cells (VSMCs) and mitochondria, and are correlated with age-associated aortic stiffness in hypercholesterolemic mice. The present study investigated whether young mice (4 months-old) with increased mitochondrial NOX4 levels recapitulate vascular aging and age-associated aortic stiffness. We generated transgenic mice with low (Nox4TG605; 2.1-fold higher) and high (Nox4TG618; 4.9-fold higher) mitochondrial NOX4 expression. Young Nox4TG618 mice showed significant increase in aortic stiffness and decrease in phenylephrine-induced aortic contraction, but not Nox4TG605 mice. Increased mitochondrial oxidative stress increased intrinsic VSMC stiffness, induced aortic extracellular matrix remodeling and fibrosis, a leftward shift in stress-strain curves, decreased volume compliance and focal adhesion turnover in Nox4TG618 mice. Nox4TG618 VSMCs phenocopied other features of vascular aging such as increased DNA damage, increased premature and replicative senescence and apoptosis, increased proinflammatory protein expression and decreased respiration. Aortic stiffening in young Nox4TG618 mice was significantly blunted with mitochondrial-targeted catalase overexpression. This demonstration of the role of mitochondrial oxidative stress in aortic stiffness will galvanize search for new mitochondrial-targeted therapeutics for treatment of age-associated vascular dysfunction.

 

 

 

 

Oxidative stress, mitochondrial dysfunction and aging are associated with the pathogenesis of cardiovascular diseases [1]. The two major reactive oxygen species (ROS) generating sources within cells are mitochondria and nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) [2,3]. Mitochondria generate ROS as a byproduct of oxidative phosphorylation, while the major function of NADPH oxidases is generation of ROS. The NOX family members include NOX1-5 and DUOX1-2 isoforms [4]. Transmembrane NOX1, NOX2, NOX4, and NOX5 catalytic subunits are expressed in vascular tissue; however, NOX5 is absent in rodents [4]. All NOX subunits, with the exception of NOX5, interact with another transmembrane protein, p22phox, forming a membrane-bound catalytic core [5]. Unlike NOX1-3, NOX4 does not require conventional cytosolic regulatory subunits for activity, but DNA polymerase-δ-interacting protein 2 was shown to stimulate NOX4 activity in vascular smooth muscle cells (VSMCs) [6]. NOX4 activity is regulated at the expression level in response to a wide range of stimuli, generating predominantly H2O2, which can diffuse through membranes and affect intra- and intercellular signaling pathways [[7], [8], [9]]. Furthermore, NOX4 localizes to mitochondria, increasing mitochondrial ROS levels [[10], [11], [12]].

 

NOX4 is present in many cell types and is highly expressed in vascular wall cells [13]; its expression and activity are significantly increased in endothelial and smooth muscle cells in response to proinflammatory mediators and cytokines [14,15]. We recently showed that NOX4 expression is increased in VSMC mitochondria and vasculature and is associated with increased aortic stiffening and atherosclerosis in aged hypercholesterolemic mice [12]. Furthermore, increased NOX4 expression in arterial wall in humans is correlated with age and atherosclerotic lesion severity [12,16].

 

Increased stiffness of large arteries, a major determinant of pulse pressure, is a significant and independent predictor of adverse cardiovascular events [17]. Conducting arteries such as aorta and carotid artery have large amount of elastin, allowing the vessel to expand and recoil to dampen the oscillatory changes in blood pressure arising from regular ventricular contractions [18]. The pressure waves, when reaching vascular branch points, are reflected back to the heart. In healthy individuals the reflected wave arrives during diastole, aiding in coronary blood flow; however, in individuals with aortic stiffening the pressure wave returns much quicker and arrives in systole, augmenting the afterload, inducing left ventricular hypertrophy, and reducing coronary blood flow. The resultant insufficiency in myocardial perfusion may cause myocardial ischemia and heart failure [19]. Furthermore, aortic stiffness is a precursor of hypertension [20].

 

Increased oxidative stress is implicated in decreased aortic compliance in patients with coronary artery disease [21]. We and others have shown that increased mitochondrial oxidative stress is a contributing factor in age-associated arterial stiffening [12,22,23]. However, it is not known whether mitochondrial oxidative stress plays a causative role in aortic stiffening. The present study was designed to determine whether increasing mitochondrial ROS production in young mice, by increasing NOX4 expression, would recapitulate the age-associated aortic stiffening observed by our laboratory and others. Our data demonstrate that increased mitochondrial oxidative stress in young mice induces aortic stiffening and VSMC phenotype similar to that observed in aged mice. Specifically, decreased aortic compliance with increased mitochondrial oxidative stress mainly results from structural remodeling of the vascular wall mediated by VSMCs rather than endothelial cells.

 

 

1. Results

 

Generation of Mitochondrial-specific Nox4 Transgenic Mice. The function of NOX4 in the pathogenesis of cardiovascular disease (CVD) is controversial. Some investigators reported that NOX4 provides protection against atherosclerosis [24] and cardiac dysfunction in response to pressure overload, particularly using young mice [25]. On the other hand, we and others showed association of increased NOX4 expression/activity with atherosclerosis [15,16,26] and cardiomyocyte hypertrophy, cell death, and cardiac fibrosis during pressure overload [27] and in cardiac remodeling [28]. In contrast to a report that NOX4 protein was not detectable in cardiomyocyte mitochondria under physiological conditions [29], at least 18 publications reported NOX4 localization to mitochondria in several cell types, including vascular cells, under pathophysiological conditions (for example [[10], [11], [12]]). We previously showed increased mitochondrial NOX4 expression in the aortic wall and VSMCs isolated from aortas with aging and its association with aortic stiffening and atherosclerosis in hypercholesterolemic mice [12].

 

Therefore, to determine whether increased mitochondrial NOX4 expression has a causative role in aging-associated aortic stiffness, we generated mitochondria-targeted Nox4 transgenic (Nox4TG) mice with varying levels of Nox4 expression, using a plasmid construct containing mouse Nox4 gene (Fig. 1A). Southern analysis of genomic DNA identified a lower (TG605) and a higher (TG618) Nox4 transgene containing founders (Fig. 1B). Gene expression analysis, measured by q-PCR, in VSMCs showed a 2-fold (Nox4TG605) and 20-fold (Nox4TG618) increase in Nox4 mRNA levels relative to the wild-type (Fig. 1C). The Nox4 expression in Nox4TG618 mice are in line with NOX4 expression in aged human carotid arteries [12]. NOX4 protein expression in liver mitochondria was increased 2.1- and 4.9-fold in Nox4TG605 and Nox4TG618, respectively, compared with the wild-type (Fig. 1D). Further supporting the increased NOX4 mitochondrial localization in Nox4TG mice, a significant increase in yellow fluorescence was observed in VSMC mitochondria from the colocalization of MitoTracker Green FM and immunoreactive NOX4 (Fig. 1 E and F).

 

 

 

Fig. 1. Generation and characterization of mitochondrial-specific Nox4 transgenic mice. (A) Essential features of the full length Nox4 overexpressing construct are depicted in the cartoon. The Nox4 gene is preceded by a Chicken β-actin promoter and a mitochondrial targeting sequence (B) Southern blot analysis for estimation of Nox4 transgene copy number in Nox4TG605 (low expresser) and Nox4TG618 (high expresser). Relative Nox4 gene copy number was assessed by spiking the wild-type genomic DNA with 0.2, 2, 20 or 200 ng of the transfection plasmid containing full length Nox4 gene. (C) Real-time reverse transcription polymerase chain reaction analysis of Nox4 mRNA expression in aortic VSMCs (mean ± SEM, N = 6). (D) Western blot analysis of NOX4 protein levels from liver mitochondria of the transgenic mice along with mitochondrial marker protein prohibitin (left panel). Densitometric quantification of NOX4 levels normalized to prohibitin levels (right panel; mean ± SEM, N = 4) (E). Representative confocal microscopy images of VSMCs showing NOX4 localization to mitochondria in wild-type, TG605, and TG618 mice. Cells were stained with MitoTracker Green FM and rabbit anti-NOX4 and AlexaFluor 647 rabbit anti-goat IgG. Bright yellow fluorescence indicates increased NOX4 levels in mitochondria. (F). Data presented was integrated density of NOX4 and MTG fluorescence (mean ± SEM, N = 30). Scale is 10 μm *P < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

 

 

Nox4TG grew normally and did not show any gross phenotypic abnormalities. Nox4TG mice were not different from wild-type littermates in appearance, body weight (25.1 ± 0.76 vs 25.36 ± 1.15 g (mean ± SEM) wild-type littermates, ~4 months-old), behavior or fertility. The inheritance of Nox4TG allele in TG605 and TG618 mice was approximately 50% in accordance with Mendelian ratios, suggesting single chromosome integration of the transgene. Nox4 transgene was expressed in all tissues studied. The expression of other NADPH oxidases ̶ Nox1 and Nox2 ̶ was not affected by Nox4 transgene expression (data not shown).

 

 

Young Nox4TG Mice Phenocopy Age-associated Aortic Stiffening and Impaired Vasomotor Function. NOX4 is a critical mediator of increased mitochondrial oxidative stress during aging [12] and mitochondrial stress/dysfunction contributes to large elastic artery stiffening and impaired contractile function [12,22,23,30]. To determine whether young Nox4TG mice will recapitulate aging-associated vascular dysfunction, we first measured pulse wave velocity (PWV). Both young Nox4TG605 and TG618 mice (4 months-old) showed an increase in PWV, but the difference was not significant between Nox4TG605 and wild-type mice (Fig. 2A). However, PWV increased 4.5-fold in Nox4TG618 mice compared with age-matched wild-type mice (Fig. 2A). PWV was also significantly higher in Nox4TG618 mice compared with age-matched Nox4TG605 mice (P < 0.05). Vasomotor function analysis of abdominal aortic rings by wire myography showed a lower but not a significant decrease in phenylephrine-induced maximum contraction in Nox4TG605 (Fig. 2B) and a 57% decrease in Nox4TG618 (Fig. 2C; P < 0.05) mice versus wild-type mice. Consistent with this, dose response curve showed that the response to phenylephrine was similar between wild-type and Nox4TG605 mice (Fig. 2D), whereas Nox4TG618 mice showed impaired aortic contraction compared with the wild-type mice (Fig. 2E; P < 0.05). However, endothelium-dependent relaxation to acetylcholine after pre-contraction with phenylephrine (SI Appendix, Fig. S1 A and B) and endothelium-independent relaxation to sodium nitroprusside (Fig. S1 C and D) were not affected in young Nox4TG mice.

 

 

 

Fig. 2. Young (4-month old) Nox4 transgenic mice with high mitochondrial NOX4 expression show increased aortic stiffness and reduced contractile function in aorta and VSMCs. (A) Pulse wave velocity (PWV) was measured in wild-type, Nox4TG605, and Nox4TG618 mice (mean ± SEM, N = 5–8; *P < 0.05 versus Nox4TG605, ***P < 0.001 versus wild-type). Maximal contractile force in abdominal aortic rings in response to phenylephrine (PE, 3.16 × 10−5 M) was measured by wire myography in (B) wild-type and Nox4TG605 and (C) wild-type and Nox4TG618 mice (mean ± SEM, N = 7; *P < 0.05). The force generation dose-response curves for PE were shown for (D) wild-type and Nox4TG605 and (E) wild-type and Nox4TG618 aortic rings (mean ± SEM, N = 7). (F and G) VSMC contraction was measured using collagen gel-based contraction assay kit and the area of the dislodged gel was measured using NIH image J software (mean ± SEM; n = 6; *P < 0.05).

 

 

We performed collagen gel contraction assay to determine whether the decrease in maximum contractile force in response to phenylephrine is partly contributed by NOX4-induced changes in the contractile phenotype of aortic SMCs [31]. Gel contraction mediated by aortic VSMCs from wild-type and Nox4TG605 was similar (Fig. 2 F and G). In comparison, the reduction in gel contraction was significantly attenuated with aortic VSMCs isolated from Nox4TG618 mice (Fig. 2 F and G), indicating that increase in mitochondrial NOX4 expression levels induces VSMC stiffening.

 

 

Increase in Intrinsic Stiffness of Aortic VSMCs Contributes to Aortic Stiffening in Young Nox4 Transgenic Mice. To further explore the underlying mechanism for the reduction in gel contraction observed with Nox4TG618 VSMCs and also because aortic stiffness with aging is attributed to intrinsic changes in VSMCs [32], we determined single VSMC stiffness using atomic force microscopy. As shown in Fig. 3, elastic modulus, a measure of stiffness, was significantly higher in aortic VSMCs from young Nox4TG618 (21.0 ± 1.3 kPa, 10 cells, 150 measurements) compared with cells from young wild-type mice (13.0 ± 0.66 kPa, 10 cells, 105 measurements), further supporting the notion that mice with increased mitochondrial NOX4 expression phenocopy vascular aging.

 

 

 

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