Heme oxygenase-1 ameliorates oxidative stress-induced endothelial senescence via regulating endothelial nitric oxide synthase activation and coupling

Results

Upregulation of HO-1 by Hemin or HO-1 adenovirus ameliorated H₂O₂-induced endothelial senescence

To investigate the effect of HO-1 on endothelial senescence, the expression of HO-1 in HUVECs was induced by the pharmacological inducer Hemin or by the recombinant adenovirus encoding HO-1 with a GFP (Ad-HO-1). As shown in Fig. 1A, the expression of HO-1 was significantly elevated by Hemin and by Ad-HO-1 infection. Endothelial senescence was induced by 50 μM H₂O₂ treated for 1 h, followed by incubation with complete culture medium for 2 days. As indicated by Fig. 1B and C, H₂O₂ significantly enhanced the proportion of senescence-associated-β-Galactosidase (SA-β-gal)-positive cells, and increased the cell size as observed by the cell morphology, suggesting that endothelial senescence was successfully induced by H₂O₂. Treatment of Hemin alone did not alter the senescent level of the HUVECs, but reversed H₂O₂-induced endothelial senescence in a dose-dependent manner (Fig. 1B). Additionally, infection of Ad-HO-1 significantly prevented H₂O₂-induced senescence of the HUVECs (Fig. 1C).

Figure 1. Induction or overexpression of HO-1 reversed HUVEC senescence. (A) Upregulation of HO-1 protein expression in HUVEC induced by Hemin or transfected with HO-1 recombinant adenovirus (HO-1, 32kDa; GFP-HO-1, 59kDa). ^*P < 0.05 vs. Control; ^#P < 0.05 vs. Ad-GFP. n = 5. (B) HUVEC were stained for SA-β-galactosidase treated with H₂O₂ (50 μM, 1 h) or/and Hemin (0.1-10 μM). Senescent cells appeared blue (200 × magnification). ^*P < 0.05 vs. Control; ^#P < 0.05 vs. H₂O₂. n = 5. (C) SA-β-gal staining of HUVEC treated with H₂O₂ (50 μM, 1 h) or/and HO-1 recombinant adenovirus (200 × magnification). ^*P < 0.05 vs. Control; ^#P < 0.05 vs. Ad-GFP+H₂O₂. n = 5. (D) Hemin attenuated the expression of p53 or p21 stimulated by H₂O₂. ^*P < 0.05 vs. Control; ^#P < 0.05 vs. H₂O₂. n = 5. (E) Overexpression of HO-1 infected by recombinant adenovirus decreased the upregulation of p53 or p21 stimulated by H₂O₂. ^*P < 0.05 vs. Ad-GFP; ^#P < 0.05 vs. Ad-GFP+H₂O₂. n = 5. (F) Hemin attenuated the expression of γH2A.X (Ser139) stimulated by H₂O₂. ^*P < 0.05 vs. Control; and ^#P < 0.05 vs. H₂O₂. n = 5.

The cyclin-dependent kinase inhibitors p53 and p21, which lead to inhibition of the cyclin E-CDK2 complex, is regarded as important mechanism underlying endothelial senescence [2]. Indeed, H₂O₂ significantly upregulated the expressions of p53 and p21 (Fig. 1D and E). The upregulation of these senescent markers was attenuated by Hemin treatment or HO-1 adenovirus infection (Fig. 1D and E). Thus, the suppression of p53 and p21 by HO-1 is probably the mechanism by which HO-1 ameliorated oxidative stress-induced endothelial aging.

Oxidative stress-induced premature endothelial senescence is also characterized by DNA damage and telomere dysfunction [27]. At the sites of DNA damage and dysfunctional telomeres, the histone variant H2A.X (γH2A.X) is phosphorylated to form DNA damage foci [28,29]. In H₂O₂-induced senescent cells, the expression of phosphorylated γH2A.X was augmented (Fig. 1F). Induction of HO-1 by Hemin reverted H₂O₂-induced increase of γH2A.X phosphorylation (Fig. 1F), suggesting that HO-1 is able to protect against DNA damage and telomere dysfunction in premature endothelial senescence.

Taken together, these observations indicate that HO-1 ameliorates oxidative stress-induced endothelial senescence.

HO-1 silencing or HO-1 inhibitor ZnPP exacerbated endothelial senescence

The effect of endogenous HO-1 on endothelial senescence was also elucidated by silencing the HO-1 gene. As shown in Fig. 2A, among the four tested siRNAs for HO-1, siRNA-3 demonstrated the best efficiency and thus used for the following studies. Knockdown of endogenous HO-1 markedly increased SA-β-gal-positive HUVECs, implying an exacerbation of endothelial premature aging (Fig. 2B). Similarly, HO inhibitor protoporphyrin IX zinc (II) (ZnPP) dose-dependently induced endothelial senescence, as implied by SA-β-gal staining (Fig. 2C). Moreover, knockdown of endogenous HO-1 enhanced the expressions of senescent markers p53 and p21 (Fig. 2D), as well as the marker of DNA damage- γH2A.X phosphorylation (Fig. 2F). ZnPP, at the concentration of 10 μM, augmented expressions of p53 and p21 (Fig. 2E), indicating that the regulation of endothelial senescence is at least partially dependent on the activity of HO-1.

Figure 2. Endogenous depletion or suppression of HO-1 promoted HUVEC senescence. (A) Screening of HO-1 interference sequences by Western blotting. Sequence-3 was used for the following experiments. ^*P < 0.05 vs. Control or NC-siRNA. n = 3. (B) SA-β-gal staining of HUVEC transfected with HO-1 siRNA (200 × magnification). ^*P < 0.05 vs. Control or NC-siRNA. n = 5. (C) SA-β-gal staining after treating with different concentration of ZnPP (0.1, 1 or 10 μM) (200 × magnification). ^*P < 0.05 vs. Control. n = 5. (D) Silencing of HO-1 increased the expression of p53 or p21. ^*P < 0.05 vs. Control or NC-siRNA. n = 5. (E) HO-1 inhibitor ZnPP increased the expression of p53 or p21. ^*P < 0.05 vs. Control. n = 5. (F) Silencing of HO-1 increased the expression of γH2A.X(Ser139). ^*P < 0.05 vs. Control or NC-siRNA. n = 5.

HO-1 increased the production of NO in senescent HUVECs

Impairment of NO production is a hallmark of endothelial aging [30–34]. In the present study, DAF-FM probe was used to detect endothelial NO production [35]. As shown in Fig. 3A, the fluorescence intensity of NO was reduced by H₂O₂ treatment, but was reversed by Hemin treatment. Contrarily, silencing of HO-1 decreased fluorescence intensity of DAF-FM (Fig. 3B). NO production was also measured according to Griess assay. As shown in Fig. 3C, Hemin reversed the reduction of endothelial NO production stimulated by H₂O₂, while this protective effect was abrogated by HO-1 inhibitor ZnPP or eNOS inhibitor N^ω-nitro-l-arginine methyl ester (L-NAME). Moreover, SA-β-galactosidase staining results showed that ZnPP or L-NAME abolished the anti-senescent effect of Hemin (Fig. 3D). It’s thus suggested that HO-1 is able to preserve the reduction of NO production in endothelial senescence, which depends on eNOS activation.

Figure 3. HO-1 advanced endothelial NO production to resist senescence in HUVECs. (A) HO-1 inducer Hemin reversed the reduction of the relative fluorescence intensity of DAF-FM indicated endothelial NO production stimulated by H₂O₂ (200 × magnification). The fluorescence intensity of DAF-FM was normalized to the cell numbers by normalizing to DAPI fluorescence (not shown). ^*P < 0.05 vs. Control; and ^#P < 0.05 vs. H₂O₂. n = 5. (B) Silencing of HO-1 decreased the relative fluorescence intensity of DAF-FM indicated endothelial NO production (200 × magnification). The fluorescence intensity of DAF-FM was normalized to the cell numbers by normalizing to DAPI fluorescence (not shown). ^*P < 0.05 vs. Control or NC-siRNA. n = 5. (C) NO production was measured according to Griess assay. Hemin reversed the reduction of endothelial NO production stimulated by H₂O₂, while the protective effect was abolished by ZnPP or L-NAME. ^*P < 0.05 vs. Control; ^#P < 0.05 vs. H₂O₂; ^$P < 0.05 vs. H₂O₂+Hemin. n = 5. (D) SA-β-galactosidase staining results showed that ZnPP or L-NAME abolished the anti-senescence effect of Hemin (200 × magnification). ^*P < 0.05 vs. Control; ^#P < 0.05 vs. H₂O₂; ^$P < 0.05 vs. H₂O₂+Hemin. n = 5.

HO-1 potentiated eNOS phosphorylation at serine 1177 via Akt

The activation of eNOS, resulting in sustaining generation of NO, is crucial for maintaining the endothelial function. The activation of eNOS is characterized by phosphorylation at serine 1177 and dephosphorylation at threonine 495 [36–38]. In senescent HUVECs induced by H₂O₂, the phosphorylation level of eNOS at Ser1177 was significantly decreased, but the total expression level of the protein was not altered (Fig. 4A and B). Both the HO-1 inducer Hemin and infection with Ad-HO-1 rendered the rebound of the phosphorylation of Ser1177 (Fig. 4A and B). By contrast, silencing of endogenous HO-1 inhibited eNOS phosphorylation at Ser1177, without affecting expression of total eNOS (Fig. 4C). These results indicate that HO-1 prevents eNOS inactivation in aging endothelial cells, and that HO-1 insufficiency triggers premature senescence. Phosphorylation of Thr495, the negative regulatory site of eNOS, was unchanged in H₂O₂-induced senescent endothelial cells, and was also unaltered by Hemin or HO-1 siRNA (Supplementary Fig. 1).

Figure 4. HO-1 retarded H₂O₂-induced HUVEC senescence through eNOS activation via Akt. (A) Expressions of p-eNOS(Ser1177) and total eNOS were measured in HUVECs treated with or without H₂O₂ and Hemin in the presence or absence of Akt inhibitor MK-2206, PKA inhibitor H-89 or AMPK inhibitor Compound C (CP-C). ^*P < 0.05 vs. Control; ^#P < 0.05 vs. H₂O₂; ^$P < 0.05 vs. H₂O₂+Hemin; n.s., non significant. n = 5. (B) Expressions of p-eNOS(Ser1177) and total eNOS were measured in HUVECs infected with or without HO-1 recombinant adenovirus in the presence or absence of Akt inhibitor MK-2206. ^*P < 0.05 vs. Ad-GFP; ^#P < 0.05 vs. Ad-GFP+H₂O₂; ^$P < 0.05 vs. Ad-HO-1+H₂O₂; n.s., non significant. n = 5. (C) Expressions of p-eNOS(Ser1177) and total eNOS were measured in HUVECs transfected with or without HO-1 siRNA. ^*P < 0.05 vs. Control or NC-siRNA; n.s., non significant. n = 5.

The phosphorylation status of a protein depends on the balanced actions of protein kinases and phosphatases. Many kinases have been identified to phosphorylate eNOS at a number of consensus motifs, including protein kinases A (PKA) [39], B (Akt) [40], and C [41], AMP-activated kinase (AMPK) [42,43], calmodulin kinase II [44]. Among all, the Phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway is the predominant upstream kinases responsible to eNOS phosphorylation at serine 1177 residue [45,46]. As shown in Fig. 4A, the restored phosphorylation of Ser1177 by Hemin was abolished by Akt inhibitor MK-2206, rather than PKA inhibitor H-89 or AMPK inhibitor Compound C. Besides, MK-2206 reversed the restored phosphorylation of Ser1177 by Ad-HO-1 (Fig. 4B), supporting the conclusion that HO-1 enhances eNOS activation via an Akt-dependent manner. However, treatment with Hemin did not change either the phosphorylation status of Akt at threonine 473 and threonine 308, or the total expression of Akt (Supplementary Fig. 2), implying that HO-1 does not directly alter Akt activation or expression.

Reportedly, protein phosphatase 2A (PP2A) has been known to dephosphorylate eNOS at serine 1177 and ultimately inhibit the activity of eNOS [47,48]. Nevertheless, in our experiments, neither the expression nor the activity of PP2A was altered by HO-1 inducer Hemin or HO-1 silencing (Supplementary Fig. 3). These observations thus suggest that the augmented eNOS phosphorylation at serine 1177 by HO-1 is not attributed to the alterations of PP2A.

HO-1 enhanced interactions between eNOS and Akt

Since HO-1 did not affect the expression or activity of the key kinase Akt, it is hypothesized that the interaction of Akt and eNOS is enhanced by HO-1. Co-IP experiments suggested that HO-1 and eNOS had physical interactions. Overexpression of HO-1 promoted the interaction between HO-1 and eNOS, as well as the interaction between eNOS and Akt in senescent HUVECs induced by H₂O₂ (Fig. 5A and B). Similarly, immunofluorescence assay confirmed that Hemin enhanced the co-localization between HO-1 and eNOS, as well as the co-localization between eNOS and Akt in senescent HUVECs induced by H₂O₂ (Fig. 5C). These observations raised the possibility that HO-1 might promote the activity of Akt enhancing interaction between Akt with eNOS.

Figure 5. HO-1 regulated endothelial senescence induced by H₂O₂ through HO-1-eNOS-Akt interaction. (A) Overexpression of HO-1 induced by Hemin enhanced the interaction between HO-1 and eNOS, and promoted the interaction between eNOS and Akt in senescent HUVECs induced by H₂O₂. ^*P < 0.05, Hemin+H₂O₂ vs. Hemin. n = 5. (B) Overexpression of HO-1 mediated by HO-1 recombinant adenovirus enhanced the interaction between HO-1 and eNOS, and promoted the interaction between eNOS and Akt in senescent HUVECs induced by H₂O₂. ^*P < 0.05, Ad-HO-1+H₂O₂ vs. Ad-HO-1. n = 5. (C) Overexpression of HO-1 enhanced the colocalization between HO-1 and eNOS, and promoted the colocalization between eNOS and Akt (400 × magnification) in senescent HUVECs induced by H₂O₂.

HO-1 prevented eNOS uncoupling

The formation of eNOS dimer is required for the normal function of eNOS, whereas uncoupled eNOS produces peroxynitrite (ONOO⁻) rather than NO [38,49]. To investigate the effect of HO-1 on eNOS coupling, the expression of eNOS dimer and monomer were compared. As shown in Fig. 6A and B, the ratio of eNOS dimer to monomer was reduced in aging cells induced by oxidative stress, but was overturned after the treatment of Hemin or HO-1 adenovirus. In contrary, knockdown of endogenous HO-1 potentiated eNOS uncoupling, as implied by the decreased ratio of eNOS dimer to monomer (Fig. 6C). Furthermore, Hemin or HO-1 adenovirus reduced the content of intracellular ROS stimulated by H₂O₂, while silencing of HO-1 increased ROS generation (Fig. 6D, E and F). Taken together, these observations indicate that HO-1 may prevent eNOS uncoupling by inhibiting ROS generation in endothelial senescence.

Figure 6. HO-1 protected the coupling state of eNOS in endothelial senescence. (A) HO-1 inducer Hemin increased the downregulation of dimer/monomer-eNOS ratio stimulated by H₂O₂. ^*P < 0.05 vs. Control; and ^#P < 0.05 vs. H₂O₂. n = 6. (B) Overexpression of HO-1 infected by recombinant adenovirus increased the downregulation of dimer/monomer-eNOS ratio stimulated by H₂O₂. ^*P < 0.05 vs. Ad-GFP; and ^#P < 0.05 vs. Ad-GFP+H₂O₂. n = 3. (C) Silencing of HO-1 decreased the dimer/monomer-eNOS ratio. ^*P < 0.05 vs. Control or NC-siRNA. n = 5. (D, E and F) Images of DHE fluorescence staining taken by confocal microscopy showing ROS production in HUVECs (200 × magnification). The fluorescence intensity of DHE was normalized to the cell numbers by normalizing to DAPI fluorescence (not shown). (D) treated with or without H₂O₂ and Hemin. ^*P < 0.05 vs. Control; and ^#P < 0.05 vs. H₂O₂. n = 4. (E) infected with or without HO-1 recombinant adenovirus. ^*P < 0.05 vs. Ad-GFP; and ^#P < 0.05 vs. Ad-GFP+H₂O₂. n = 6. (F) transfected with or without HO-1 siRNA. ^*P < 0.05 vs. Control or NC-siRNA. n = 6.

HO-1 alleviated in vivo endothelial senescence in the aorta of SHRs

In SHRs, endothelial premature senescence and dysfunction are caused by a consistently elevated arterial blood pressure [50]. Thus, SHR is considered to a typical in vivo model of endothelial senescence. 12-week-old SHRs received a 10-day treatment of Hemin to observe the in vivo effect of HO-1. The age-matched WKYs were served as the normotensive control group, while the untreated SHRs were served as the model group. At the end of treatment, all the animals were sacrificed, and the aortas were isolated for SA-β-gal staining and Western blot analysis. Upregulation of HO-1 induced by Hemin reduced blood pressure in the SHRs (Fig. 7A). As shown in Fig. 7B, the aortas of SHRs had significantly higher SA-β-gal blue-staining level compared to WKY controls. In aortas of Hemin-treated SHRs, a significant decrease in SA-β-gal staining signal was observed as compared to that of the untreated-SHRs. Additionally, Hemin treatment decreased the expression of p53 in SHRs (Fig. 7C). As shown in Fig. 7D, Hemin reversed the downregulation of p-eNOS (Ser1177) in SHRs, while didn’t affect the expression of total eNOS. Consistently with the in vitro experiments, these results confirm that HO-1 prevents endothelial senescence through regulating eNOS.

Figure 7. HO-1 alleviated endothelial senescence through regulating phosphorylation status of eNOS at Ser1177 in SHRs. (A) Upregulation of HO-1 reduced blood pressure in the SHR. ^*P < 0.05 vs. WKY; ^#P < 0.05 vs. SHR. n = 6. (B) SA-β-gal staining of the aortas originated from WKYs, SHRs, and SHRs treated with Hemin. (C) Hemin attenuated the high expression of p53 in the SHR. ^*P < 0.05 vs. WKY; ^#P < 0.05 vs. SHR. n = 8. (D) Hemin increased the downregulation of p-eNOS (Ser1177) in SHRs, while had no effect on the expression of eNOS. ^*P < 0.05 vs. WKY; ^#P < 0.05 vs. SHR; n.s., non significant. n = 7-12.

Discussion

Premature senescence of vascular endothelial cells induced by oxidative stress is a hallmark of the decline of endothelial function, and has been proposed to be involved in the development of various cardiovascular diseases such as hypertension and atherosclerosis [1,2,51–53]. Therapeutic strategies targeting endothelial senescence have shed new lights on the treatment of cardiovascular diseases [7]. The present study revealed that HO-1 ameliorates H₂O₂-induced premature endothelial senescence and is considered to be a promising target. This conclusion is firstly supported by the observations that treatment with HO-1 inducer Hemin or infection of HO-1 adenovirus prevented H₂O₂-induced expression of cell senescence marker SA-β-gal, upregulation of cell cycle inhibitor p53 and p21, as well as increase of phosphorylated γH2A.X level which indicates DNA damage and telomere dysfunction. Additionally, the observations that depletion of endogenous HO-1 by siRNA or treatment with ZnPP, the competitive inhibitor of HO-1 which inhibits HO activity with or without altering its expression [54], enhanced expressions of these senescent markers, further confirm that deficiency of endogenous HO-1 promoted endothelial senescence and that the effect of HO-1 is at least partially dependent on its activity. In line with the findings that activation of the Nrf2-dependent cellular anti-oxidant defense system prevents endothelial senescence (Supplementary Fig. 4) [55–57], and that inhibition of Nrf2 significantly promotes cellular senescence [58,59], these findings suggest that HO-1, as one of the most important downstream target genes of Nrf2, contributes to the anti-senescent effects of Nrf2.

Mechanistically, the effect of HO-1 on endothelial senescence is most likely due to its regulation in NO production. Indeed, the impairment of NO release and decrease of NO bioavailability have been implicated in senescent endothelial cells [31–34,51]. Treatment with NO donor DETA-NO or transfection with eNOS protects against the development of endothelial senescence [60–62], whereas the eNOS inhibitor L-NAME or Asymmetrical dimethylarginine (ADMA) ab [56] abrogates the anti-senescent effect of NO and elicits endothelial and vascular senescence [60,61,63,64]. In this study, NO production detected by DAF-FM fluorescent probe and Griess assay was significantly enhanced by Hemin treatment, but demonstrated a reduction by HO-1 knockdown, suggesting that HO-1 is able to influence the generation of NO. In addition, L-NAME abolished the protective effect of Hemin on augmenting NO production and preventing endothelial senescence, further supporting the conclusions that the anti-senescent effect of HO-1 is attributed to its improvement in NO generation. The mechanisms underlying the regulation of NO in endothelial aging are still undetermined. Some studies indicate that NO enhances telomerase activity, as implied by the results that eNOS transfection and NO donor are comparable to hTERT transfection in increasing telomerase activity and inhibiting cellular senescence [60,62]; whereas a study provides evidence challenging that physiological concentrations of NO could modulate telomerase levels [65]. Additionally, it has been suggested that defective eNOS activity, which leads to a gradual reduction of NO bioavailability but an increased formation of harmful free radicals including peroxynitrite (ONOO⁻) and N₂O₃ in the vasculature, causes DNA and telomere damage to facilitate endothelial aging [66,67]. The damage of genomic stability is characterized by the phosphorylation of the γH2A.X to form DNA damage foci, the stabilization of p53 through phosphorylation, the increase in the expression of its downstream transcriptional target p21, and finally inhibition of the cyclin E-CDK2 complex [2]. Thus, the observations of the present study that upregulation or overexpression of HO-1 diminished oxidative stress-induced phosphorylation of γH2A.X and expressions of p53 and p21, meanwhile HO-1 deficiency or inhibition trigger these responses of DNA and telomere damage, might suggest that eNOS activation by HO-1 is probably involved in its anti-senescent properties.

Since the activation of eNOS results from the phosphorylation of the enzyme [37,38,68], the phosphorylation level of eNOS was firstly determined to find out the answer how HO-1 modulates eNOS activation and NO production. The most important phosphorylation sites of eNOS are serine 1177 and threonine 495. The former can increase the Ca²⁺ sensitivity and represent an additional and independent mechanism of eNOS activation; while the latter is a negative regulatory site, the phosphorylation of which can interfere with the binding of calmodulin [37,38]. In the presence of the essential cofactor tetrahydrobiopterin (BH4), the activated eNOS oxidizes its substrate L-arginine to form NO and L-citrulline [69]. A major finding of our study is that HO-1 potentiated eNOS phosphorylation at Ser1177 to enhance eNOS activation. This conclusion is supported by the results that Hemin treatment or Ad-HO-1 infection reversed the decreased eNOS phosphorylation at Ser1177 induced by H₂O₂ while HO-1 silencing restrained this phosphorylation, and that HO-1 did not alter either the total expression of eNOS nor the phosphorylation at Thr495. Nevertheless, HO-1 is not a kinase or phosphatase, so it seems impossible that HO-1 activates eNOS directly. Among all the kinases and phosphatases responsible to eNOS phosphorylation at Ser1177, Akt is the predominant kinase [45] and PP2A is the most important phosphatase [47,48]. Thus, we detected the activation of Akt and PP2A in HUVECs with HO-1 induction or knockdown. Our work showed that HO-1 did not affect either the phosphorylation of Akt at Ser473 or Thr308, the total expression of Akt, nor the activity or expression of PP2A. Therefore, it would rule out the possibility that HO-1 influences eNOS activation through altering its upstream kinases or phosphatases. Surprisingly, we discovered that there was a protein-protein interaction between HO-1 and eNOS, as implied by the co-IP and immunofluorescence results. Overexpression of HO-1 enhanced the co-localization and interaction of HO-1 and eNOS, and advanced the interaction between eNOS and Akt. We speculate that the binding of HO-1 to eNOS might alter the space conformation of eNOS, enhancing its interaction with Akt, subsequently facilitating the phosphorylation of eNOS by Akt. In fact, similar interactions between heat shock protein 90 (Hsp90) and eNOS or nNOS have been identified, which could activate the NOSs by modulating their phosphorylation or binding with Ca²⁺/calmodulin [70–72]. Since HO-1 is also regarded as a heat shock protein 32 (Hsp32), for its promoter regions containing heat shock elements similar to those identified in the regulatory regions of various heat shock protein genes [73], it is possible that HO-1 shares conformational and functional similarity with Hsp90 in the regulation of the NOS activity.

In addition, uncoupling of eNOS is an important mechanism of oxidative stress and decreased NO bioavailability in endothelial and vascular senescence [27,74,75]. In the aging endothelial cells, increased amount of ·O₂⁻ reacts with NO to form ONOO⁻, which leads to eNOS uncoupling via oxidation of BH4 [74,76]. The uncoupled eNOS shifts the nitroso-redox balance favoring production of ·O₂⁻ rather than NO, resulting in further increased formation of endothelial ROS and the activation of redox-sensitive genes that contribute to endothelial dysfunction [74,76–79]. Indeed, our experiments demonstrated eNOS uncoupling in senescent HUVEC model induced by H₂O₂. Interestingly, HO-1 induction or overexpression reduced the formation of uncoupled eNOS, whereas silencing of endogenous HO-1 triggered eNOS uncoupling. Since HO-1 is a potent antioxidant enzyme against ROS, as demonstrated by the DHE fluorescence experiments, it is most likely that HO-1 diminished ROS production to prevent eNOS uncoupling in endothelial senescence.

The effect of HO-1 on endothelial senescence was also investigated in vivo by using SHRs as a model. In SHRs, chronic high blood pressure induces endothelial dysfunction, as demonstrated by decrease of NO bioavailability, impairment of endothelium-dependent hyperpolarization, and facilitation of endothelium-dependent contractions [1,80,81]. NO donor exerted anti-hypertensive effects in SHRs through the restoration of vascular endothelial protective functions [82]. Our findings demonstrated that endothelium of aortas collected from 12-week-old SHRs presented a severe senescent phenotype, as implied by SA-β-gal staining results and p53 expression level, and as compared with the age-matched normotensive control WKYs. Treatment of HO-1 pharmacological inducer Hemin for 10 days ameliorated endothelial senescence in SHRs aortas. In addition, induction of HO-1 alleviated endothelial senescence through regulating phosphorylation status of eNOS at Ser1177. The present findings are in line with the in vitro results and confirm that induction/overexpression of HO-1 prevents endothelial aging induced by hypertension. Notably, upregulation of HO-1 lowers arterial blood pressure in the SHRs [17–21]. The inhibitory effect of HO-1 against endothelial senescence via regulating eNOS might contribute to its improvement of endothelial function and anti-hypertensive properties. Interestingly, the mechanisms by which HO-1 protects against endothelial dysfunction seem to differ from different stages of hypertension. In 12 weeks old SHRs which are used in the present study, HO-1 mainly enhances eNOS activation and inhibits eNOS uncoupling to reverse endothelial premature aging; whereas in 36 weeks old SHRs, up-regulation of HO-1 reverses endothelial dysfunction via impairing endothelium-dependent contractions and enhancing endothelium-dependent hyperpolarization, without affecting NO-mediated vasodilatations [20,21]. This discrepancy might probably due to the dysfunction of eNOS and impaired responsiveness of sGC to NO in the old SHRs [83–85].

Intriguingly, as an inducible enzyme, the expression of HO-1 was induced during the development of endothelial aging resulted from H₂O₂ treatment (Supplementary Fig. 5A). Taken into account that HO-1 exerts protective effects against endothelial senescence, this induction of HO-1 might be an intracellular compensatory mechanism against oxidative stress-induced endothelial aging. However, it is important to note that compensatory induction of HO-1 was not sustained and was attenuated 24 hours after H₂O₂ stimulation (Supplementary Fig. 5A). Moreover, the expression of HO-1 was observed to be decreased in the senescent HUVECs (Passage 11 of sub-cultured cells) as compared with the young cells (Passage 3) (Supplementary Fig. 5B). These observations thus suggest that the induction of HO-1 as an endogenous cellular compensation against senescence is limited. Pharmacological inducers of HO-1 or gene therapy delivery of HO-1 gene, which lead to sustained induction or upregulation of HO-1, might be more promising for therapeutic use. Since most of the currently available pharmacological inducers of HO-1, in particular the heavy metals, show cellular and tissue toxicity [86], there is a need to develop new HO-1 inducers that are effective and safe. Natural compounds which show a potent effect on HO-1 induction or enhancement of the upstream Nrf-2/HO-1 pathway may be promising sources. Moreover, the application of genetic interventions may allow selective tissue targeting of HO-1 induction, and may lead to the development of more effective and long-lasting therapies for cardiovascular diseases such as hypertension and its vascular complications.

In summary, the present study suggests that HO-1 ameliorates oxidative stress-induced premature senescence in HUVECs via regulating eNOS through the following mechanisms: 1) HO-1 interacts with eNOS to enhance the interaction between eNOS and Akt, and then improves eNOS phosphorylation at Ser1177; 2) HO-1 provides antioxidant protection to endothelial cells and maintains the stable state of eNOS dimer, finally resulting in increased production of endothelial NO (Fig. 8). These findings provide important new insights into the effect of HO-1 on treating endothelial senescence and dysfunction in hypertension or other cardiovascular diseases.

Figure 8. HO-1 ameliorates H₂O₂-induced premature senescence in HUVECs via eNOS. HO-1 attenuates H₂O₂-induced expressions of p53/p21 and ameliorated oxidative stress-induced DNA damage in endothelia senescence through two mechanisms: 1) HO-1 prevents eNOS uncoupling by inhibiting ROS. 2) Interaction between HO-1 and eNOS makes eNOS easier to interact with Akt, for which Akt can phosphorylate eNOS at Ser1177.

Acknowledgements

We thank Prof. Paul M. Vanhoutte from The University of Hong Kong for the kind advices for the study.

Conflicts of Interest

No conflicts of interest are declared by the authors.

Funding

The study is supported by National Natural Science Foundation of China (No. 81400359, 81473205, 81673433), Guangdong Natural Science Foundation (S2013040014102), Specialized Research Fund for the Doctoral Program of Higher Education of China (20130171120048), Training Program for young teachers from Sun Yat-sen University (No.15ykpy03), National Engineering and Technology Research Center for New drug Druggability Evaluation (Seed Program of Guangdong Province), Major Project of Guangdong Provincial Department of Science and Technology (2015B020232009, 2014B020210003 and 2013B090700010), Major Project of Platform Construction Education Department of Guangdong Province (No. 2014GKPT002), Guangzhou Science and Technology Program Project (No. 201604020121), and funding from Guangdong Provincial Key Laboratory of Construction Foundation (No. 2017B030314030).

References

• 1. Félétou M, Vanhoutte PM. Endothelial dysfunction: a multifaceted disorder (The Wiggers Award Lecture). Am J Physiol Heart Circ Physiol. 2006; 291:H985–1002. https://doi.org/10.1152/ajpheart.00292.2006 [PubMed]
• 2. Erusalimsky JD. Vascular endothelial senescence: from mechanisms to pathophysiology. J Appl Physiol (1985). 2009; 106:326–32. https://doi.org/10.1152/japplphysiol.91353.2008 [PubMed]
• 3. Brown MF, Stuart JA. Correlation of mitochondrial superoxide dismutase and DNA polymerase beta in mammalian dermal fibroblasts with species maximal lifespan. Mech Ageing Dev. 2007; 128:696–705. https://doi.org/10.1016/j.mad.2007.10.004 [PubMed]
• 4. Erusalimsky JD, Kurz DJ. Cellular senescence in vivo: its relevance in ageing and cardiovascular disease. Exp Gerontol. 2005; 40:634–42. https://doi.org/10.1016/j.exger.2005.04.010 [PubMed]
• 5. Davignon J, Ganz P. Role of endothelial dysfunction in atherosclerosis. Circulation. 2004 (Suppl 1); 109:III27–32. https://doi.org/10.1161/01.CIR.0000131515.03336.f8 [PubMed]
• 6. Deanfield JE, Halcox JP, Rabelink TJ. Endothelial function and dysfunction: testing and clinical relevance. Circulation. 2007; 115:1285–95. https://doi.org/10.1161/CIRCULATIONAHA.106.652859 [PubMed]
• 7. Naylor RM, Baker DJ, van Deursen JM. Senescent cells: a novel therapeutic target for aging and age-related diseases. Clin Pharmacol Ther. 2013; 93:105–16. https://doi.org/10.1038/clpt.2012.193 [PubMed]
• 8. Park SY, Kwon OS, Andtbacka RH, Hyngstrom JR, Reese V, Murphy MP, Richardson RS. Age-related endothelial dysfunction in human skeletal muscle feed arteries: the role of free radicals derived from mitochondria in the vasculature. Acta Physiol (Oxf). 2018; 222 https://doi.org/doi.org/10.1111/apha.12893 [PubMed]
• 9. Converso DP, Taillé C, Carreras MC, Jaitovich A, Poderoso JJ, Boczkowski J. HO-1 is located in liver mitochondria and modulates mitochondrial heme content and metabolism. FASEB J. 2006; 20:1236–38. https://doi.org/10.1096/fj.05-4204fje [PubMed]
• 10. Alam J, Stewart D, Touchard C, Boinapally S, Choi AM, Cook JL. Nrf2, a Cap’n’Collar transcription factor, regulates induction of the heme oxygenase-1 gene. J Biol Chem. 1999; 274:26071–78. https://doi.org/10.1074/jbc.274.37.26071 [PubMed]
• 11. Tenhunen R, Marver HS, Schmid R. The enzymatic catabolism of hemoglobin: stimulation of microsomal heme oxygenase by hemin. J Lab Clin Med. 1970; 75:410–21. [PubMed]
• 12. Frydman RB, Tomaro ML, Buldain G, Awruch J, Díaz L, Frydman B. Specificity of heme oxygenase: a study with synthetic hemins. Biochemistry. 1981; 20:5177–82. https://doi.org/10.1021/bi00521a012 [PubMed]
• 13. Zhang H, Liu YY, Jiang Q, Li KR, Zhao YX, Cao C, Yao J. Salvianolic acid A protects RPE cells against oxidative stress through activation of Nrf2/HO-1 signaling. Free Radic Biol Med. 2014; 69:219–28. https://doi.org/10.1016/j.freeradbiomed.2014.01.025 [PubMed]
• 14. Yang C, Zhang X, Fan H, Liu Y. Curcumin upregulates transcription factor Nrf2, HO-1 expression and protects rat brains against focal ischemia. Brain Res. 2009; 1282:133–41. https://doi.org/10.1016/j.brainres.2009.05.009 [PubMed]
• 15. Abraham NG, Kappas A. Pharmacological and clinical aspects of heme oxygenase. Pharmacol Rev. 2008; 60:79–127. https://doi.org/10.1124/pr.107.07104 [PubMed]
• 16. Cheng C, Noordeloos AM, Jeney V, Soares MP, Moll F, Pasterkamp G, Serruys PW, Duckers HJ. Heme oxygenase 1 determines atherosclerotic lesion progression into a vulnerable plaque. Circulation. 2009; 119:3017–27. https://doi.org/10.1161/CIRCULATIONAHA.108.808618 [PubMed]
• 17. Sacerdoti D, Escalante B, Abraham NG, McGiff JC, Levere RD, Schwartzman ML. Treatment with tin prevents the development of hypertension in spontaneously hypertensive rats. Science. 1989; 243:388–90. https://doi.org/10.1126/science.2492116 [PubMed]
• 18. Ushiyama M, Morita T, Katayama S. Carbon monoxide regulates blood pressure cooperatively with nitric oxide in hypertensive rats. Heart Vessels. 2002; 16:189–95. https://doi.org/10.1007/s003800200020 [PubMed]
• 19. Wang R, Shamloul R, Wang X, Meng Q, Wu L. Sustained normalization of high blood pressure in spontaneously hypertensive rats by implanted hemin pump. Hypertension. 2006; 48:685–92. https://doi.org/10.1161/01.HYP.0000239673.80332.2f [PubMed]
• 20. Li Z, Wang Y, Vanhoutte PM. Upregulation of heme oxygenase 1 by hemin impairs endothelium-dependent contractions in the aorta of the spontaneously hypertensive rat. Hypertension. 2011; 58:926–34. https://doi.org/10.1161/HYPERTENSIONAHA.111.173807 [PubMed]
• 21. Li Z, Wang Y, Man RY, Vanhoutte PM. Upregulation of heme oxygenase-1 potentiates EDH-type relaxations in the mesenteric artery of the spontaneously hypertensive rat. Am J Physiol Heart Circ Physiol. 2013; 305:H1471–83. https://doi.org/10.1152/ajpheart.00962.2012 [PubMed]
• 22. Pertusa M, García-Matas S, Rodríguez-Farré E, Sanfeliu C, Cristòfol R. Astrocytes aged in vitro show a decreased neuroprotective capacity. J Neurochem. 2007; 101:794–805. https://doi.org/10.1111/j.1471-4159.2006.04369.x [PubMed]
• 23. Pascal T, Debacq-Chainiaux F, Boilan E, Ninane N, Raes M, Toussaint O. Heme oxygenase-1 and interleukin-11 are overexpressed in stress-induced premature senescence of human WI-38 fibroblasts induced by tert-butylhydroperoxide and ethanol. Biogerontology. 2007; 8:409–22. https://doi.org/10.1007/s10522-007-9084-8 [PubMed]
• 24. Berge U, Kristensen P, Rattan SI. Hormetic modulation of differentiation of normal human epidermal keratinocytes undergoing replicative senescence in vitro. Exp Gerontol. 2008; 43:658–62. https://doi.org/10.1016/j.exger.2007.12.009 [PubMed]
• 25. Tarry-Adkins JL, Chen JH, Smith NS, Jones RH, Cherif H, Ozanne SE. Poor maternal nutrition followed by accelerated postnatal growth leads to telomere shortening and increased markers of cell senescence in rat islets. FASEB J. 2009; 23:1521–28. https://doi.org/10.1096/fj.08-122796 [PubMed]
• 26. Kim HJ, Ham SA, Paek KS, Hwang JS, Jung SY, Kim MY, Jin H, Kang ES, Woo IS, Kim HJ, Lee JH, Chang KC, Han CW, Seo HG. Transcriptional up-regulation of antioxidant genes by PPARδ inhibits angiotensin II-induced premature senescence in vascular smooth muscle cells. Biochem Biophys Res Commun. 2011; 406:564–69. https://doi.org/10.1016/j.bbrc.2011.02.091 [PubMed]
• 27. Donato AJ, Morgan RG, Walker AE, Lesniewski LA. Cellular and molecular biology of aging endothelial cells. J Mol Cell Cardiol. 2015; 89:122–35. https://doi.org/10.1016/j.yjmcc.2015.01.021 [PubMed]
• 28. Takai H, Smogorzewska A, de Lange T. DNA damage foci at dysfunctional telomeres. Curr Biol. 2003; 13:1549–56. https://doi.org/10.1016/S0960-9822(03)00542-6 [PubMed]
• 29. Turinetto V, Giachino C. Multiple facets of histone variant H2AX: a DNA double-strand-break marker with several biological functions. Nucleic Acids Res. 2015; 43:2489–98. https://doi.org/10.1093/nar/gkv061 [PubMed]
• 30. Minamino T, Miyauchi H, Yoshida T, Ishida Y, Yoshida H, Komuro I. Endothelial cell senescence in human atherosclerosis: role of telomere in endothelial dysfunction. Circulation. 2002; 105:1541–44. https://doi.org/10.1161/01.CIR.0000013836.85741.17 [PubMed]
• 31. Versari D, Daghini E, Virdis A, Ghiadoni L, Taddei S. The ageing endothelium, cardiovascular risk and disease in man. Exp Physiol. 2009; 94:317–21. https://doi.org/10.1113/expphysiol.2008.043356 [PubMed]
• 32. Matsushita H, Chang E, Glassford AJ, Cooke JP, Chiu CP, Tsao PS. eNOS activity is reduced in senescent human endothelial cells: preservation by hTERT immortalization. Circ Res. 2001; 89:793–98. https://doi.org/10.1161/hh2101.098443 [PubMed]
• 33. Sato I, Morita I, Kaji K, Ikeda M, Nagao M, Murota S. Reduction of nitric oxide producing activity associated with in vitro aging in cultured human umbilical vein endothelial cell. Biochem Biophys Res Commun. 1993; 195:1070–76. https://doi.org/10.1006/bbrc.1993.2153 [PubMed]
• 34. Lee MY, Wang Y, Vanhoutte PM. Senescence of cultured porcine coronary arterial endothelial cells is associated with accelerated oxidative stress and activation of NFkB. J Vasc Res. 2010; 47:287–98. https://doi.org/10.1159/000265563 [PubMed]
• 35. Balcerczyk A, Soszynski M, Bartosz G. On the specificity of 4-amino-5-methylamino-2′,7′-difluorofluorescein as a probe for nitric oxide. Free Radic Biol Med. 2005; 39:327–35. https://doi.org/10.1016/j.freeradbiomed.2005.03.017 [PubMed]
• 36. Rafikov R, Fonseca FV, Kumar S, Pardo D, Darragh C, Elms S, Fulton D, Black SM. eNOS activation and NO function: structural motifs responsible for the posttranslational control of endothelial nitric oxide synthase activity. J Endocrinol. 2011; 210:271–84. https://doi.org/10.1530/JOE-11-0083 [PubMed]
• 37. Fleming I, Busse R. Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase. Am J Physiol Regul Integr Comp Physiol. 2003; 284:R1–12. https://doi.org/10.1152/ajpregu.00323.2002 [PubMed]
• 38. Förstermann U, Münzel T. Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation. 2006; 113:1708–14. https://doi.org/10.1161/CIRCULATIONAHA.105.602532 [PubMed]
• 39. Walther S, Pluteanu F, Renz S, Nikonova Y, Maxwell JT, Yang LZ, Schmidt K, Edwards JN, Wakula P, Groschner K, Maier LS, Spiess J, Blatter LA, et al. Urocortin 2 stimulates nitric oxide production in ventricular myocytes via Akt- and PKA-mediated phosphorylation of eNOS at serine 1177. Am J Physiol Heart Circ Physiol. 2014; 307:H689–700. https://doi.org/10.1152/ajpheart.00694.2013 [PubMed]
• 40. Michell BJ, Griffiths JE, Mitchelhill KI, Rodriguez-Crespo I, Tiganis T, Bozinovski S, de Montellano PR, Kemp BE, Pearson RB. The Akt kinase signals directly to endothelial nitric oxide synthase. Curr Biol. 1999; 9:845–48. https://doi.org/10.1016/S0960-9822(99)80371-6 [PubMed]
• 41. Li P, Zhang L, Zhang M, Zhou C, Lin N. Uric acid enhances PKC-dependent eNOS phosphorylation and mediates cellular ER stress: A mechanism for uric acid-induced endothelial dysfunction. Int J Mol Med. 2016; 37:989–97. https://doi.org/10.3892/ijmm.2016.2491 [PubMed]
• 42. Wang S, Miao J, Qu M, Yang GY, Shen L. Adiponectin modulates the function of endothelial progenitor cells via AMPK/eNOS signaling pathway. Biochem Biophys Res Commun. 2017; 493:64–70. https://doi.org/10.1016/j.bbrc.2017.09.073 [PubMed]
• 43. Liu Y, Oh SJ, Chang KH, Kim YG, Lee MY. Antiplatelet effect of AMP-activated protein kinase activator and its potentiation by the phosphodiesterase inhibitor dipyridamole. Biochem Pharmacol. 2013; 86:914–25. https://doi.org/10.1016/j.bcp.2013.07.009 [PubMed]
• 44. Saura M, Marquez S, Reventun P, Olea-Herrero N, Arenas MI, Moreno-Gómez-Toledano R, Gómez-Parrizas M, Muñóz-Moreno C, González-Santander M, Zaragoza C, Bosch RJ. Oral administration of bisphenol A induces high blood pressure through angiotensin II/CaMKII-dependent uncoupling of eNOS. FASEB J. 2014; 28:4719–28. https://doi.org/10.1096/fj.14-252460 [PubMed]
• 45. Wei Q, Xia Y. Proteasome inhibition down-regulates endothelial nitric-oxide synthase phosphorylation and function. J Biol Chem. 2006; 281:21652–59. https://doi.org/10.1074/jbc.M602105200 [PubMed]
• 46. García-Morales V, Luaces-Regueira M, Campos-Toimil M. The cAMP effectors PKA and Epac activate endothelial NO synthase through PI3K/Akt pathway in human endothelial cells. Biochem Pharmacol. 2017; 145:94–101. https://doi.org/10.1016/j.bcp.2017.09.004 [PubMed]
• 47. Greif DM, Kou R, Michel T. Site-specific dephosphorylation of endothelial nitric oxide synthase by protein phosphatase 2A: evidence for crosstalk between phosphorylation sites. Biochemistry. 2002; 41:15845–53. https://doi.org/10.1021/bi026732g [PubMed]
• 48. Wu Y, Song P, Xu J, Zhang M, Zou MH. Activation of protein phosphatase 2A by palmitate inhibits AMP-activated protein kinase. J Biol Chem. 2007; 282:9777–88. https://doi.org/10.1074/jbc.M608310200 [PubMed]
• 49. Schulz E, Jansen T, Wenzel P, Daiber A, Münzel T. Nitric oxide, tetrahydrobiopterin, oxidative stress, and endothelial dysfunction in hypertension. Antioxid Redox Signal. 2008; 10:1115–26. https://doi.org/10.1089/ars.2007.1989 [PubMed]
• 50. Vanhoutte PM, Feletou M, Taddei S. Endothelium-dependent contractions in hypertension. Br J Pharmacol. 2005; 144:449–58. https://doi.org/10.1038/sj.bjp.0706042 [PubMed]
• 51. Minamino T, Miyauchi H, Yoshida T, Ishida Y, Yoshida H, Komuro I. Endothelial cell senescence in human atherosclerosis: role of telomere in endothelial dysfunction. Circulation. 2002; 105:1541–44. https://doi.org/10.1161/01.CIR.0000013836.85741.17 [PubMed]
• 52. Voghel G, Thorin-Trescases N, Farhat N, Nguyen A, Villeneuve L, Mamarbachi AM, Fortier A, Perrault LP, Carrier M, Thorin E. Cellular senescence in endothelial cells from atherosclerotic patients is accelerated by oxidative stress associated with cardiovascular risk factors. Mech Ageing Dev. 2007; 128:662–71. https://doi.org/10.1016/j.mad.2007.09.006 [PubMed]
• 53. Vanhoutte PM, Shimokawa H, Tang EH, Feletou M. Endothelial dysfunction and vascular disease. Acta Physiol (Oxf). 2009; 196:193–222. https://doi.org/10.1111/j.1748-1716.2009.01964.x [PubMed]
• 54. Morioka I, Wong RJ, Abate A, Vreman HJ, Contag CH, Stevenson DK. Systemic effects of orally-administered zinc and tin (IV) metalloporphyrins on heme oxygenase expression in mice. Pediatr Res. 2006; 59:667–72. https://doi.org/10.1203/01.pdr.0000215088.71481.a6 [PubMed]
• 55. Wang RY, Liu LH, Liu H, Wu KF, An J, Wang Q, Liu Y, Bai LJ, Qi BM, Qi BL, Zhang L. Nrf2 protects against diabetic dysfunction of endothelial progenitor cells via regulating cell senescence. Int J Mol Med. 2018. https://doi.org/10.3892/ijmm.2018.3727 [PubMed]
• 56. Senthil KK, Gokila VM, Wang SY. Activation of Nrf2-mediated anti-oxidant genes by antrodin C prevents hyperglycemia-induced senescence and apoptosis in human endothelial cells. Oncotarget. 2017; 8:96568–87. https://doi.org/10.18632/oncotarget.19951 [PubMed]
• 57. Yang G, Zhao K, Ju Y, Mani S, Cao Q, Puukila S, Khaper N, Wu L, Wang R. Hydrogen sulfide protects against cellular senescence via S-sulfhydration of Keap1 and activation of Nrf2. Antioxid Redox Signal. 2013; 18:1906–19. https://doi.org/10.1089/ars.2012.4645 [PubMed]
• 58. Volonte D, Liu Z, Musille PM, Stoppani E, Wakabayashi N, Di YP, Lisanti MP, Kensler TW, Galbiati F. Inhibition of nuclear factor-erythroid 2-related factor (Nrf2) by caveolin-1 promotes stress-induced premature senescence. Mol Biol Cell. 2013; 24:1852–62. https://doi.org/10.1091/mbc.e12-09-0666 [PubMed]
• 59. Valcarcel-Ares MN, Gautam T, Warrington JP, Bailey-Downs L, Sosnowska D, de Cabo R, Losonczy G, Sonntag WE, Ungvari Z, Csiszar A. Disruption of Nrf2 signaling impairs angiogenic capacity of endothelial cells: implications for microvascular aging. J Gerontol A Biol Sci Med Sci. 2012; 67:821–29. https://doi.org/10.1093/gerona/glr229 [PubMed]
• 60. Hayashi T, Matsui-Hirai H, Miyazaki-Akita A, Fukatsu A, Funami J, Ding QF, Kamalanathan S, Hattori Y, Ignarro LJ, Iguchi A. Endothelial cellular senescence is inhibited by nitric oxide: implications in atherosclerosis associated with menopause and diabetes. Proc Natl Acad Sci USA. 2006; 103:17018–23. https://doi.org/10.1073/pnas.0607873103 [PubMed]
• 61. Zhong W, Zou G, Gu J, Zhang J. L-arginine attenuates high glucose-accelerated senescence in human umbilical vein endothelial cells. Diabetes Res Clin Pract. 2010; 89:38–45. https://doi.org/10.1016/j.diabres.2010.03.013 [PubMed]
• 62. Vasa M, Breitschopf K, Zeiher AM, Dimmeler S. Nitric oxide activates telomerase and delays endothelial cell senescence. Circ Res. 2000; 87:540–42. https://doi.org/10.1161/01.RES.87.7.540 [PubMed]
• 63. Boe AE, Eren M, Murphy SB, Kamide CE, Ichimura A, Terry D, McAnally D, Smith LH, Miyata T, Vaughan DE. Plasminogen activator inhibitor-1 antagonist TM5441 attenuates Nω-nitro-L-arginine methyl ester-induced hypertension and vascular senescence. Circulation. 2013; 128:2318–24. https://doi.org/10.1161/CIRCULATIONAHA.113.003192 [PubMed]
• 64. Scalera F, Borlak J, Beckmann B, Martens-Lobenhoffer J, Thum T, Täger M, Bode-Böger SM. Endogenous nitric oxide synthesis inhibitor asymmetric dimethyl L-arginine accelerates endothelial cell senescence. Arterioscler Thromb Vasc Biol. 2004; 24:1816–22. https://doi.org/10.1161/01.ATV.0000141843.77133.fc [PubMed]
• 65. Hong Y, Quintero M, Frakich NM, Trivier E, Erusalimsky JD. Evidence against the involvement of nitric oxide in the modulation of telomerase activity or replicative capacity of human endothelial cells. Exp Gerontol. 2007; 42:904–10. https://doi.org/10.1016/j.exger.2007.01.007 [PubMed]
• 66. Bautista-Niño PK, Portilla-Fernandez E, Vaughan DE, Danser AH, Roks AJ. DNA Damage: A Main Determinant of Vascular Aging. Int J Mol Sci. 2016; 17:17. https://doi.org/10.3390/ijms17050748 [PubMed]
• 67. Kumar RG, Spurthi MK, Kumar KG, Sahu SK, Rani SH. Endothelial nitric oxide synthase polymorphism G298T in association with oxidative DNA damage in coronary atherosclerosis. J Genet. 2012; 91:349–52. https://doi.org/10.1007/s12041-012-0183-1 [PubMed]
• 68. Schini-Kerth VB, Vanhoutte PM. Nitric oxide synthases in vascular cells. Exp Physiol. 1995; 80:885–905. https://doi.org/10.1113/expphysiol.1995.sp003904 [PubMed]
• 69. Michel T, Feron O. Nitric oxide synthases: which, where, how, and why? J Clin Invest. 1997; 100:2146–52. https://doi.org/10.1172/JCI119750 [PubMed]
• 70. Park JH, Kim WS, Kim JY, Park MH, Nam JH, Yun CW, Kwon YG, Jo I. Chk1 and Hsp90 cooperatively regulate phosphorylation of endothelial nitric oxide synthase at serine 1179. Free Radic Biol Med. 2011; 51:2217–26. https://doi.org/10.1016/j.freeradbiomed.2011.09.021 [PubMed]
• 71. Song Y, Zweier JL, Xia Y. Heat-shock protein 90 augments neuronal nitric oxide synthase activity by enhancing Ca2+/calmodulin binding. Biochem J. 2001; 355:357–60. https://doi.org/10.1042/bj3550357 [PubMed]
• 72. Song Y, Zweier JL, Xia Y. Determination of the enhancing action of HSP90 on neuronal nitric oxide synthase by EPR spectroscopy. Am J Physiol Cell Physiol. 2001; 281:C1819–24. https://doi.org/10.1152/ajpcell.2001.281.6.C1819 [PubMed]
• 73. Kiemer AK, Bildner N, Weber NC, Vollmar AM. Characterization of heme oxygenase 1 (heat shock protein 32) induction by atrial natriuretic peptide in human endothelial cells. Endocrinology. 2003; 144:802–12. https://doi.org/10.1210/en.2002-220610 [PubMed]
• 74. Brandes RP, Fleming I, Busse R. Endothelial aging. Cardiovasc Res. 2005; 66:286–94. https://doi.org/10.1016/j.cardiores.2004.12.027 [PubMed]
• 75. Rajapakse AG, Yepuri G, Carvas JM, Stein S, Matter CM, Scerri I, Ruffieux J, Montani JP, Ming XF, Yang Z. Hyperactive S6K1 mediates oxidative stress and endothelial dysfunction in aging: inhibition by resveratrol. PLoS One. 2011; 6:e19237. https://doi.org/10.1371/journal.pone.0019237 [PubMed]
• 76. Förstermann U, Sessa WC. Nitric oxide synthases: regulation and function. Eur Heart J. 2012; 33:829–37, 837a–837d. https://doi.org/10.1093/eurheartj/ehr304 [PubMed]
• 77. Montezano AC, Touyz RM. Reactive oxygen species and endothelial function--role of nitric oxide synthase uncoupling and Nox family nicotinamide adenine dinucleotide phosphate oxidases. Basic Clin Pharmacol Toxicol. 2012; 110:87–94. https://doi.org/10.1111/j.1742-7843.2011.00785.x [PubMed]
• 78. Csiszar A, Ungvari Z, Edwards JG, Kaminski P, Wolin MS, Koller A, Kaley G. Aging-induced phenotypic changes and oxidative stress impair coronary arteriolar function. Circ Res. 2002; 90:1159–66. https://doi.org/10.1161/01.RES.0000020401.61826.EA [PubMed]
• 79. Charles S, Raj V, Arokiaraj J, Mala K. Caveolin1/protein arginine methyltransferase1/sirtuin1 axis as a potential target against endothelial dysfunction. Pharmacol Res. 2017; 119:1–11. https://doi.org/10.1016/j.phrs.2017.01.022 [PubMed]
• 80. Vanhoutte PM, Boulanger CM. Endothelium-dependent responses in hypertension. Hypertens Res. 1995; 18:87–98. https://doi.org/10.1291/hypres.18.87 [PubMed]
• 81. Vanhoutte PM, Shimokawa H, Feletou M, Tang EH. Endothelial dysfunction and vascular disease - a 30th anniversary update. Acta Physiol (Oxf). 2017; 219:22–96. https://doi.org/10.1111/apha.12646 [PubMed]
• 82. Monti M, Ciccone V, Pacini A, Roggeri R, Monzani E, Casella L, Morbidelli L. Anti-hypertensive property of a nickel-piperazine/NO donor in spontaneously hypertensive rats. Pharmacol Res. 2016; 107:352–59. https://doi.org/10.1016/j.phrs.2016.03.033 [PubMed]
• 83. Zhou XJ, Vaziri ND, Zhang J, Wang HW, Wang XQ. Association of renal injury with nitric oxide deficiency in aged SHR: prevention by hypertension control with AT1 blockade. Kidney Int. 2002; 62:914–21. https://doi.org/10.1046/j.1523-1755.2002.00516.x [PubMed]
• 84. Vaziri ND, Wang XQ, Ni ZN, Kivlighn S, Shahinfar S. Effects of aging and AT-1 receptor blockade on NO synthase expression and renal function in SHR. Biochim Biophys Acta. 2002; 1592:153–61. https://doi.org/10.1016/S0167-4889(02)00309-9 [PubMed]
• 85. Ndisang JF, Wang R. Alterations in heme oxygenase/carbon monoxide system in pulmonary arteries in hypertension. Exp Biol Med (Maywood). 2003; 228:557–63. https://doi.org/10.1177/15353702-0322805-27 [PubMed]
• 86. Peterson SJ, Frishman WH, Abraham NG. Targeting heme oxygenase: therapeutic implications for diseases of the cardiovascular system. Cardiol Rev. 2009; 17:99–111. https://doi.org/10.1097/CRD.0b013e31819d813a [PubMed]