Red Blood Cell and Endothelial eNOS Independently Regulate Circulating Nitric Oxide Metabolites and Blood Pressure

Abstract

Background: Current paradigms suggest that nitric oxide (NO) produced by endothelial cells (ECs) through endothelial nitric oxide synthase (eNOS) in the vessel wall is the primary regulator of blood flow and blood pressure. However, red blood cells (RBCs) also carry a catalytically active eNOS, but its role is controversial and remains undefined. This study aimed to elucidate the functional significance of RBC eNOS compared with EC eNOS for vascular hemodynamics and nitric oxide metabolism.

Methods: We generated tissue-specific loss- and gain-of-function models for eNOS by using cell-specific Cre-induced gene inactivation or reactivation. We created 2 founder lines carrying a floxed eNOS (eNOS^flox/flox) for Cre-inducible knockout (KO), and gene construct with an inactivated floxed/inverted exon (eNOS^inv/inv) for a Cre-inducible knock-in (KI), which respectively allow targeted deletion or reactivation of eNOS in erythroid cells (RBC eNOS KO or RBC eNOS KI mice) or in ECs (EC eNOS KO or EC eNOS KI mice). Vascular function, hemodynamics, and nitric oxide metabolism were compared ex vivo and in vivo.

Results: The EC eNOS KOs exhibited significantly impaired aortic dilatory responses to acetylcholine, loss of flow-mediated dilation, and increased systolic and diastolic blood pressure. RBC eNOS KO mice showed no alterations in acetylcholine-mediated dilation or flow-mediated dilation but were hypertensive. Treatment with the nitric oxide synthase inhibitor N^γ-nitro-l-arginine methyl ester further increased blood pressure in RBC eNOS KOs, demonstrating that eNOS in both ECs and RBCs contributes to blood pressure regulation. Although both EC eNOS KOs and RBC eNOS KOs had lower plasma nitrite and nitrate concentrations, the levels of bound NO in RBCs were lower in RBC eNOS KOs than in EC eNOS KOs. Reactivation of eNOS in ECs or RBCs rescues the hypertensive phenotype of the eNOS^inv/inv mice, whereas the levels of bound NO were restored only in RBC eNOS KI mice.

Conclusions: These data reveal that eNOS in ECs and RBCs contribute independently to blood pressure homeostasis.

Keywords: blood circulation; blood pressure; hypertension; models, animal; nitric oxide synthase.

Figure 1.

Generation and characterization of EC eNOS KO or RBC eNOS KO mice. A, Scheme describing the gene-targeting strategy showing the position of the loxP sequences (black) within the gene-targeting construct before and after exon 2 of Nos3 was used to generate the founder eNOS^flox/flox mice. B, To generate EC eNOS KO mice and their respective WT control (eNOS^flox/flox Cdh5-Cre/ERT2^neg), the founder eNOS^flox/flox mice were crossed with endothelial-specific tamoxifen (TAM)-inducible Cdh5-Cre/ERT2^pos mice to obtain eNOS^flox/flox Cdh5-Cre/ERT2^pos/neg mice; these were treated with TAM for 5 days and analyzed after 21 days. To create RBC eNOS KO mice or global eNOS KO mice and their respective WT littermate controls, the founder eNOS^flox/flox was crossed with erythroid-specific (Hbb-Cre^pos) mice or with Deleter-Cre^pos mice (expressing Cre in all tissues). C, Real-time PCR analysis shows that tissue-specific DNA recombination occurs in the aorta of EC eNOS KO mice (blue), whereas in RBC eNOS KO mice, DNA recombination occurs in the bone marrow, but not in the aorta (orange). No recombination is observed in WT littermate control mice (white). t test **P<0.01, ****P<0.0001 vs respective WT control. D, Top, Real-time reverse transcriptase PCR analysis shows that endothelial cells (CD31⁺ CD45⁻) extracted from the lung of EC eNOS KO mice (blue) have a significant loss of eNOS expression compared with WT controls. *Mann-Whitney U test P=0.0286. Instead, the expression of eNOS in lung EC (CD31⁺ CD45⁻) from RBC eNOS KO (orange) is not different from WT control. It is notable that TAM increased the expression of eNOS in lung EC from WT (eNOS^flox/floxCdh5-Cre/ERT2^neg) mice. Bottom, Cre recombinase is expressed in lung endothelial cells from EC eNOS KO (blue) but not in endothelial cells from WT (white) or RBC eNOS KO (orange) mice. *Mann-Whitney U test P=0.0286. E, Top, Real-time reverse transcriptase PCR analysis shows loss of eNOS expression and Cre recombinase expression in the aorta of EC eNOS KO (blue), but not in WT littermate control mice (white). TAM increased mRNA eNOS expression in the aorta of the WT (eNOS^flox/floxCdh5-Cre/ERT2^neg) mice, but the protein levels are not different from WT mice (see also I, ELISA). One-way ANOVA P<0.0001; Tukey multiple comparison test **P<0.01,*** P<0.001, ****P<0.0001 vs respective WT control. F, Top, Immunoblot (IB) analysis shows loss of eNOS (135 kDa) expression in the aorta of EC eNOS KO (eNOS^flox/floxCdh5-Cre/ERT2^pos+TAM) mice but not in littermate controls (eNOS^flox/floxCdh5-Cre/ERT2^neg+TAM) after treatment with TAM. Loading control actin (45 kDa). Note: Sample in the first lane shows a residue of eNOS expression in that particular mouse because the knockdown efficiency of this model is ≈90% to 95%. Bottom, Immunoblot of aorta of RBC eNOS KO mice and WT littermate controls, showing that eNOS expression is not different in these 2 groups. G, Real-time reverse transcriptase PCR analysis shows eNOS expression and Cre recombinase expression in the bone marrow of RBC eNOS KO mice. Cre-recombinase is expressed in the bone marrow of RBC eNOS KO mice, but not in the aorta. Note that, although eNOS deletion was specifically detected in the bone marrow of RBC eNOS KO mice, we determined similar eNOS expression levels in the RBC eNOS KO mice compared with WT littermates; this is attributable to the high abundance of endothelial cells in the bone marrow. ****P<0.0001 Mann-Whitney U test vs WT mice. H, Upper, Immunoblot of membrane preparations of RBCs (ghosts) showing the presence of eNOS in WT RBCs and its absence in RBCs from RBC eNOS KO mice. Bottom, Immunoprecipitation (IP) of eNOS (135 KDa) from RBC lysates shows eNOS expression in WT mice and lack of eNOS in global eNOS KO mice (gKO) and RBC eNOS KO mice. The IgG (120 kDa) is seen in the IP samples. I, Electron scanning microscopy with immunogold staining of eNOS in WT (eNOS^flox/flox HbbCre^pos) mice (Upper) and RBC eNOS KO (Bottom). J, The quantification of eNOS protein expression in multiple organs by ELISA shows a significant loss of eNOS in multiple tissues of EC eNOS KO mice compared with WT littermate controls. The differences between the WT control groups are not significant, except in the spleen. Data were analyzed according to mixed-effect model with Geisser-Greenhouse correction (variables: strain, tissue); P<0.0001; multiple comparisons of eNOS levels among the groups within the same tissue were assessed by a Tukey test **P<0.01; ***P<0.001; ****P<0.000. Lines represent means± SD. EC indicates endothelial cell; eNOS, endothelial nitric oxide synthase; KO, knockout; MWM, molecular weight marker; RBC, red blood cell; and WT, wild type.

Figure 2.

Vascular endothelial dilator function is lost in EC eNOS KO mice and preserved in RBC eNOS KO mice. Nitric oxide–dependent vascular endothelial function is fully abolished in EC eNOS KO mice and fully preserved in RBC eNOS KO mice compared with the respective littermate controls. A, Preconstricted aortic rings from EC eNOS KO lack acetylcholine (ACh)-induced vasodilation (2-way repeated measurement [RM]-ANOVA P<0.0001; Sidak ***P<0.001 vs WT control for concentrations of ACh>10^–6.5 mol/L (n= 6 per group). B, Flow-mediated dilation (FMD) of the iliac artery assessed in vivo by ultrasound is abolished in EC eNOS KO (blue; 2-way RM-ANOVA P<0.0001; Sidak **P<0.01 vs WT control [white] for t>6.3 minutes; n=10 per group). C, In aortic rings from RBC eNOS KO (orange), ACh-induced vasodilation was not different from WT littermates (white). Two-way RM-ANOVA P<0.0001. n=5 per group. D, FMD of the iliac artery is fully preserved in RBC eNOS KO mice, 2-way RM-ANOVA P<0.0001. n=8 per group. E, Endothelium-dependent relaxation (EDR) in response to ACh (calculated as the percentage of the maximal ACh response) is significantly impaired in EC eNOS KO and fully preserved in RBC eNOS KO compared with their respective WT controls. One-way ANOVA P<0.0001; Tukey test ****P<0.0001. F, Maximal FMD (corresponding to the percentage of maximal flow-mediated dilator response) is significantly decreased in EC eNOS KO mice and fully preserved in RBC eNOS KO mice, compared with their respective WT controls. Lines represent means±SD. One-way ANOVA P<0.0001; Tukey test ***P<0.001. EC indicates endothelial cell; eNOS, endothelial nitric oxide synthase; KO, knockout; RBC, red blood cell; PCR, polymerase chain reaction; and WT, wild type.

Figure 3.

RBC eNOS and EC eNOS both contribute to blood pressure homeostasis. A, Invasive measurements of blood pressure (BP) in anesthetized mice show that both EC eNOS KO and RBC eNOS KO mice have increased mean arterial pressure (MAP) compared with their respective WT littermate controls. Global eNOS KO mice (gKO=eNOS^flox/flox Deleter Cre) are hypertensive and show significantly higher MAP than EC eNOS KO and RBC eNOS KO mice. One-way ANOVA P<0.001; Tukey *P<0.05; ****P<0.0001. B, Systemic vascular resistance (SVR) was estimated in a subcohort of mice by measuring MAP and cardiac output (CO) by ultrasound in the same animal (SVR ≈ MAP/CO) and was significantly increased in both EC eNOS KO and RBC eNOS KO mice compared with their respective controls. One-way-ANOVA P=0.0149; Welch t test P<0.05. Inset Top, The heart rate (HR) depicted here represents the values measured by cardiac ultrasound. Inset Bottom, Cardiac output. See also Table 2 for other cardiac parameters. C, Mean of MAP measurements performed in awake EC eNOS KO (n=7) and WT littermates (n=5) by radiotelemetry showing the diurnal and nocturnal variation of blood pressure with highest values during the night. KO (SD = ±10 mm Hg) vs WT (SD = ±8 mm Hg); Welch t test *P<0.001. Refer also to Figure VII in the Data Supplement for mean diurnal values. D, Mean of MAP measurements performed in awake RBC eNOS KO (n=8) and WT littermates (n=8) by radiotelemetry. KO (SD = ±15 mm Hg) vs WT (SD = ±13 mm Hg); Welch t test correction *P<0.001. Refer also to Figure VII in the Data Supplement for mean diurnal values. E, Telemetric measurements of changes in systolic BP (SBP) in awake EC eNOS KO mice (n=7) show that NOS inhibition by L-NAME or increase in arginine availability by the administration of the arginase inhibitor NorNOHA did not affect SBP in awake EC eNOS KO mice (blue) compared with WT mice (n=5). Two-way RM ANOVA P<0.001; Holm-Sidak *P<0.05 vs baseline. See Figure IV in the Data Supplement for data on diastolic BP and HR. F, Radiotelemetric measurements of changes in SBP in a subcohort of awake RBC eNOS KO mice (n=5) show that treatment with L-NAME further increases SBP in RBC eNOS KO and WT littermate (n=3) to the same extent as in the WT controls (although the baseline levels between RBC eNOS KO and WT controls were significantly different, see C and D, this figure); increase of arginine bioavailability by the administration of NorNOHA rapidly restored BP to the baseline levels. Two-way RM ANOVA P<0.001; Holm-Sidak *P<0.05 vs baseline. BL indicates baseline; EC, endothelial cell; eNOS, endothelial nitric oxide synthase; i.p., intraperitoneally; KO, knockout; L-NAME, N^γ-nitro-l-arginine methyl ester; NorNOHA, N-hydroxy-nor-l-arginine; p.o., orally; RBC, red blood cell; RM, repeated measures; TAM, tamoxifen; and WT, wild type.

Figure 4.

Reactivation of eNOS expression in either ECs or RBCs rescues global eNOS KO mice from hypertension. A, Scheme describing the gene-targeting strategy showing the position of a loxP (black) and loxP511 (green) sequences within the gene-targeting construct used to generate eNOS^inv/inv mice, which are conditional eNOS KO mice (CondKO). B, Schematic representation of the crossing strategy. To create EC eNOS KI mice (green), eNOS^inv/inv mice=CondKO mice (black) were crossed with endothelial-specific tamoxifen-inducible Cre mouse (Cdh5-Cre/ERT2^pos) and treated with tamoxifen for 5 days and analyzed after 21 days. To create RBC eNOS KI mice (yellow), eNOS^inv/inv mice=CondKO mice (black) were crossed with erythroid-specific (HbbCre^pos) mice. C, Real-time polymerase chain reaction analysis shows that tissue-specific DNA recombination occurs in the aorta of EC eNOS KI mice (blue), whereas in RBC eNOS KI mice (yellow), DNA recombination occurs in the bone marrow, but not in the aorta. No DNA recombination is observed in CondKO littermate control mice (white). t test *P<0.05, ****P<0.0001 vs respective CondKO control. D, Real-time reverse transcriptase polymerase chain reaction analysis eNOS expression in the aorta of EC eNOS KI mice (green) but not in CondKO littermate control mice (black). One-way ANOVA P<0.0001; Tukey multiple comparisons test *P<0.05 vs respective CondKO control. E, Representative immunoblot analysis demonstrating eNOS expression after Cre-dependent reactivation of eNOS expression in aorta EC eNOS KI mice, compared with CondKO, EC eNOS KO, RBC KO, and WT controls. Refer also to J (eNOS expression by ELISA) and Figure IX in the Data Supplement for immunoblot analysis from other tissues. F, Representative Western blot analysis demonstrating lack of eNOS expression in the aorta of RBC eNOS KI mice and CondKO mice. See Figure IX in the Data Supplement for immunoblot analysis from other tissues. G, Real-time reverse transcriptase polymerase chain reaction analysis shows that erythroid cells (Ter119⁺CD71⁺CD45⁻) extracted from the bone marrow of RBC eNOS KI mice (yellow, n=3) express eNOS mRNA compared with CondKO controls, where we could not detect any eNOS mRNA (n=3) *P=0.05 (exact) Mann-Whitney U test. H, ELISA detection of eNOS expression in RBC membrane preparations (ghosts) from RBC eNOS KI mice (n=4) compared with CondKO controls (n=4), where we could not detect any eNOS protein. *P=0.0286 (exact) Mann-Whitney U test. I, Low (Upper) and high (Lower) contrast images representing immunoprecipitation (IP) and immunoblot (IB) analysis of eNOS (135 kDa) from RBC lysates show eNOS expression in RBC eNOS KI mice and lack of eNOS in global eNOS KO mice (gKO) and CondKO mice. The IgG (120 kDa) is seen in the IP samples. J, The quantification of eNOS protein expression in multiple organs by ELISA shows a significant eNOS expression in multiple tissues of EC eNOS KI mice, whereas no eNOS was detected in RBC eNOS KI or littermate CondKO mice. Data were analyzed according to the mixed-effect model with Geisser-Greenhouse correction; P<0.0001; multiple comparisons of eNOS levels among the groups within the same tissue were assessed by a Tukey test; P<0.0001; Tukey *P<0.001. Lines represent means± SD. EC indicates endothelial cell; eNOS, endothelial nitric oxide synthase; KI, knock-in; KO, knockout; MWM, molecular weight marker; RBC, red blood cell; and WT, wild type.

Figure 5.

Reactivation of eNOS expression in either ECs or RBCs rescues global eNOS KO mice from hypertension. A and B, Vascular endothelial dilator function is fully restored in EC eNOS KI. A, Preconstricted aortic rings from CondKO mice (black, n=2) lack acetylcholine (ACh)-induced vasodilation, whereas reactivation of eNOS fully restores ACh response in EC eNOS KI (green, n = 3). Two-way repeated-measures ANOVA concentration P<0.0001; KI vs KO P=0.0461; Sidak **P<0.01 vs CondKO control for concentrations of ACh>10^–7 mol/L. B, Flow-mediated dilation of the iliac artery assessed in vivo by ultrasound is abolished in CondKO mice (black; 2-way repeated-measures ANOVA time P=0.0007 KI vs KO P=0.329; Fisher least significant difference test **P<0.01 vs CondKO control (black) for t >6.3 minutes); and restored in EC eNOS KI mice (green). C, Invasive blood pressure analysis shows that reactivation of eNOS expression in ECs in EC eNOS KI mice significantly decreases MAP compared with their littermate conditional eNOS KO mice (CondKO). One-way ANOVA P<0.001; Tukey ****P<0.0001. D, Systemic vascular resistance (SVR) was estimated in a subcohort of mice by measuring MAP and cardiac output (CO) by ultrasound in the same animal (SVR ≈ MAP/CO). The heart rate depicted here represents the values measured by cardiac ultrasound. SVR is decreased in EC eNOS KI compared with CondKO mice; t test Welch *P<0.05. See also Table 3 for other cardiac parameters. CondKO, conditional global eNOS knockout mice; EC, endothelial cell; eNOS, endothelial nitric oxide synthase; KI, knock-in; KO, knockout; MAP, mean arterial pressure; RBC, red blood cell; and WT, wild type.

Figure 6.

RBC eNOS and EC eNOS both contribute to plasma nitrite, but red cell eNOS is the major determinant of circulating NO-heme. A, A significant decrease in plasma nitrite is observed in both EC eNOS KO (blue) and RBC eNOS KO (orange), compared with their respective WT controls (white). Welch t test *P<0.05; **P<0.01. B, Plasma nitrite in EC eNOS KI (green) or RBC eNOS KI (yellow) is not different from CondKO mice (black), but CondKO mice show lower plasma nitrite levels than WT mice (white; see also Table 4). C, The levels of nitrosospecies (RXNO=RSNO+RNNO) were unchanged in EC eNOS KO (blue) and significantly higher in the plasma of RBC eNOS KO (orange) mice, compared with their respective WT control (white). Welch t test *P<0.05. D, The levels of RXNO in plasma of EC eNOS KI, RBC eNOS KI, and CondKO mice are comparable. E, NO-heme concentrations in RBCs were unchanged in EC eNOS KO mice and decreased in RBC eNOS KO mice. Welch t test *P<0.05. F, Accordingly, concentrations of NO-heme were unchanged in EC eNOS KI mice (green) but higher in RBC eNOS KI mice (yellow) compared with CondKO mice (black), indicating that RBC eNOS is the major determinant of circulating NO-heme. Welch t test *P<0.05. CondKO, conditional global eNOS knockout mice; EC, endothelial cell; eNOS, endothelial nitric oxide synthase; KI, knock-in; KO, knockout; NO, nitric oxide; RBC, red blood cell; RXNO, nitrosospecies (nitrosothiols+nitrosamines); and WT, wild type.