Muscle RING Finger-1 Promotes a Maladaptive Phenotype in Chronic Hypoxia-Induced Right Ventricular Remodeling

Exposure to chronic hypoxia (CH) induces elevated pulmonary artery pressure/resistance, leading to an eventual maladaptive right ventricular hypertrophy (RVH). Muscle RING finger-1 (MuRF1) is a muscle-specific ubiquitin ligase that mediates myocyte atrophy and has been shown to play a role in left ventricular hypertrophy and altered cardiac bioenergetics in pressure overloaded hearts. However, little is known about the contribution of MuRF1 impacting RVH in the setting of CH. Therefore, we hypothesized that MuRF1 deletion would enhance RVH compared to their wild-type littermates, while cardiac-specific overexpression would reduce hypertrophy following CH-induced pulmonary hypertension. We assessed right ventricular systolic pressure (RVSP), right ventricle to left ventricle plus septal weight ratio (RV/LV+S) and hematocrit (Hct) following a 3-wk isobaric CH exposure. Additionally, we conducted dual-isotope SPECT/CT imaging with cardiac function agent ²⁰¹Tl-chloride and cell death agent ^99mTc-annexin V. Predictably, CH induced pulmonary hypertension, measured by increased RVSP, RV/LV+S and Hct in WT mice compared to normoxic WT mice. Normoxic WT and MuRF1-null mice exhibited no significant differences in RVSP, RV/LV+S or Hct. CH-induced increases in RVSP were also similar between WT and MuRF1-null mice; however, RV/LV+S and Hct were significantly elevated in CH-exposed MuRF1-null mice compared to WT. In cardiac-specific MuRF1 overexpressing mice, RV/LV+S increased significantly due to CH exposure, even greater than in WT mice. This remodeling appeared eccentric, maladaptive and led to reduced systemic perfusion. In conclusion, these results are consistent with an atrophic role for MuRF1 regulating the magnitude of right ventricular hypertrophy following CH-induction of pulmonary hypertension.     

Extracellular superoxide dismutase overexpression can reverse the course of hypoxia-induced pulmonary hypertension

Hypoxia leads to free radical production, which has a pivotal role in the pathophysiology of pulmonary hypertension (PH). We hypothesized that treatment with extracellular superoxide dismutase (EC-SOD) could ameliorate the development of PH induced by hypoxia. In vitro studies using pulmonary microvascular endothelial cells showed that cells transfected with EC-SOD had significantly less accumulation of xanthine oxidase and reactive oxygen species than nontransfected cells after hypoxia exposure for 24 h. To study the prophylactic role of EC-SOD, adult male wild-type (WT) and transgenic (TG) mice, with lung-specific overexpression of human EC-SOD (hEC-SOD), were exposed to fraction of inspired oxygen (FiO(2)) 10% for 10 d. After exposure, right ventricular systolic pressure (RVSP), right ventricular mass (RV/S + LV), pulmonary vascular wall thickness (PVWT) and pulmonary artery contraction/relaxation were assessed. TG mice were protected against PH compared with WT mice with significantly lower RVSP (23.9 ± 1.24 versus 47.2 ± 3.4), RV/S + LV (0.287 ± 0.015 versus 0.335 ± 0.022) and vascular remodeling, indicated by PVWT (14.324 ± 1.107 versus 18.885 ± 1.529). Functional studies using pulmonary arteries isolated from mice indicated that EC-SOD prevents hypoxia-mediated attenuation of nitric oxide-induced relaxation. Therapeutic potential was assessed by exposing WT mice to FiO(2) 10% for 10 d. Half of the group was transfected with plasmid containing cDNA encoding human EC-SOD. The remaining animals were transfected with empty vector. Both groups were exposed to FiO(2) 10% for a further 10 d. Transfected mice had significantly reduced RVSP (18.97 ± 1.12 versus 41.3 ± 1.5), RV/S + LV (0.293 ± 0.012 versus 0.372 ± 0.014) and PVWT (12.51 ± 0.72 versus 18.98 ± 1.24). On the basis of these findings, we concluded that overexpression of EC-SOD prevents the development of PH and ameliorates established PH.     

Increased pulmonary vascular resistance and defective pulmonary artery filling in caveolin-1-/- mice

Caveolin-1, the structural and signaling protein of caveolae, is an important negative regulator of endothelial nitric oxide synthase (eNOS). We observed that mice lacking caveolin-1 (Cav1(-/-)) had twofold increased plasma NO levels but developed pulmonary hypertension. We measured pulmonary vascular resistance (PVR) and assessed alterations in small pulmonary arteries to determine the basis of the hypertension. PVR was 46% greater in Cav1(-/-) mice than wild-type (WT), and increased PVR in Cav1(-/-) mice was attributed to precapillary sites. Treatment with NG-nitro-l-arginine methyl ester (l-NAME) to inhibit NOS activity raised PVR by 42% in WT but 82% in Cav1(-/-) mice, indicating greater NO-mediated pulmonary vasodilation in Cav1(-/-) mice compared with WT. Pulmonary vasculature of Cav1(-/-) mice was also less reactive to the vasoconstrictor thromboxane A2 mimetic (U-46619) compared with WT. We observed redistribution of type I collagen and expression of smooth muscle alpha-actin in lung parenchyma of Cav1(-/-) mice compared with WT suggestive of vascular remodeling. Fluorescent agarose casting also showed markedly decreased density of pulmonary arteries and artery filling defects in Cav1(-/-) mice. Scanning electron microscopy showed severely distorted and tortuous pulmonary precapillary vessels. Thus caveolin-1 null mice have elevated PVR that is attributed to remodeling of pulmonary precapillary vessels. The elevated basal plasma NO level in Cav1(-/-) mice compensates partly for the vascular structural abnormalities by promoting pulmonary vasodilation.     

Effect of dietary sodium on vasoconstriction and eNOS-mediated vascular relaxation in caveolin-1-deficient mice

Changes in dietary sodium intake are associated with changes in vascular volume and reactivity that may be mediated, in part, by alterations in endothelial nitric oxide synthase (eNOS) activity. Caveolin-1 (Cav-1), a transmembrane anchoring protein in the plasma membrane caveolae, binds eNOS and limits its translocation and activation. To test the hypothesis that endothelial Cav-1 participates in the dietary sodium-mediated effects on vascular function, we assessed vascular responses and nitric oxide (NO)-mediated mechanisms of vascular relaxation in Cav-1 knockout mice (Cav-1-/-) and wild-type control mice (WT; Cav-1+/+) placed on a high-salt (HS; 4% NaCl) or low-salt (LS; 0.08% NaCl) diet for 16 days. After the systolic blood pressure was measured, the thoracic aorta was isolated for measurement of vascular reactivity and NO production, and the heart was used for measurement of eNOS expression and/or activity. The blood pressure was elevated in HS mice treated with NG-nitro-l-arginine methyl ester and more so in Cav-1-/- than WT mice and was significantly reduced during the LS diet. Phenylephrine caused vascular contraction that was significantly reduced in Cav-1-/- (maximum 0.25 +/- 0.06 g/mg) compared with WT (0.75 +/- 0.22 g/mg) on the HS diet, and the differences were eliminated with the LS diet. Also, vascular contraction in response to membrane depolarization by high KCl (96 mM) was reduced in Cav-1-/- (0.27 +/- 0.05 g/mg) compared with WT mice (0.53 +/- 0.12 g/mg) on the HS diet, suggesting that the reduced vascular contraction is not limited to a particular receptor. Acetylcholine (10(-5) M) caused aortic relaxation in WT mice on HS (23.6 +/- 3.5%) and LS (23.7 +/- 5.5%) that was enhanced in Cav-1-/- HS (72.6 +/- 6.1%) and more so in Cav-1-/- LS mice (93.6 +/- 3.5%). RT-PCR analysis indicated increased eNOS mRNA expression in the aorta and heart, and Western blots indicated increased total eNOS and phosphorylated eNOS in the heart of Cav-1-/- compared with WT mice on the HS diet, and the genotypic differences were less apparent during the LS diet. Thus Cav-1 deficiency during the HS diet is associated with decreased vasoconstriction, increased vascular relaxation, and increased eNOS expression and activity, and these effects are altered during the LS diet. The data support the hypothesis that endothelial Cav-1, likely through an effect on eNOS activity, plays a prominent role in the regulation of vascular function during substantial changes in dietary sodium intake.     

eNOS-deficient mice show reduced pulmonary vascular proliferation and remodeling to chronic hypoxia

Pulmonary hypertension is characterized by structural and morphological changes to the lung vasculature. To determine the potential role of nitric oxide in the vascular remodeling induced by hypoxia, we exposed wild-type [WT(+/+)] and endothelial nitric oxide synthase (eNOS)-deficient [(-/-)] mice to normoxia or hypoxia (10% O(2)) for 2, 4, and 6 days or for 3 wk. Smooth muscle alpha-actin and von Willebrand factor immunohistochemistry revealed significantly less muscularization of small vessels in hypoxic eNOS(-/-) mouse lungs than in WT(+/+) mouse lungs at early time points, a finding that correlated with decreases in proliferating vascular cells (5-bromo-2'-deoxyuridine positive) at 4 and 6 days of hypoxia in the eNOS(-/-) mice. After 3 wk of hypoxia, both mouse types exhibited similar percentages of muscularized small vessels; however, only the WT(+/+) mice exhibited an increase in the percentage of fully muscularized vessels and increased vessel wall thickness. eNOS protein expression was increased in hypoxic WT(+/+) mouse lung homogenates at all time points examined, with significantly increased percentages of small vessels expressing eNOS protein after 3 wk. These results indicate that eNOS deficiency causes decreased muscularization of small pulmonary vessels in hypoxia, likely attributable to the decrease in vascular cell proliferation observed in these mice.     

低氧性肺动脉高压小鼠肺组织apoE蛋白表达的变化及其意义

目的：观察低氧性肺动脉高压小鼠肺组织中载脂蛋白E（apoE）蛋白表达的变化,以探讨低氧性肺动脉高压形成过程中apoE蛋白表达的变化及可能的意义。方法：SPF级雄性野生型（WT）C57BL/6小鼠和雄性apoE基因敲除（apoE-KO）小鼠各20只,各随机再分为2组（n=10）：常氧组和低氧组,共4组。常压连续低氧3周（9%~11% O₂,23 h/d）复制慢性低氧性肺动脉高压模型,采用右心导管法测定小鼠右心室压（RVSP）,计算右心室与左心室加室间隔重量比RV/（LV+S）,ELISA法检测血浆中高密度脂蛋白（HDL）、低密度脂蛋白（LDL）和总胆固醇（TC）的含量;Western blot法检测肺组织中apoE和过氧化物酶体增殖物激活受体γ（PPARγ）蛋白的表达。结果：①低氧组WT小鼠RVSP、RV/（LV+S）分别较常氧组高68%和59%（P均<0.05）,血浆中HDL含量及HDL/LDL比值分别较常氧组低17%和40%（P均<0.05）,同时肺、肝组织中apoE及肺组织中PPARγ的蛋白表达分别较常氧组下调48%、52%和37%（P均<0.05）,RVSP与apoE及PPARγ蛋白表达均呈显著负相关（P均<0.01）;②低氧组apoE-KO小鼠RVSP、RV/（LV+S）较常氧组分别高96%和86%（P均<0.05）,低氧组apoE-KO小鼠RVSP和RV/（LV+S）较低氧组WT小鼠分别高29%和24%（P均<0.05）。结论：小鼠低氧性肺动脉高压的形成与肺组织中apoE蛋白表达下调有关。     

Caveolin-1-eNOS signaling promotes p190RhoGAP-A nitration and endothelial permeability

Endothelial barrier function is regulated by adherens junctions (AJs) and caveolae-mediated transcellular pathways. The opening of AJs that is observed in caveolin-1(-/-) (Cav-1(-/-)) endothelium suggests that Cav-1 is necessary for AJ assembly or maintenance. Here, using endothelial cells isolated from Cav-1(-/-) mice, we show that Cav-1 deficiency induced the activation of endothelial nitric oxide synthase (eNOS) and the generation of nitric oxide (NO) and peroxynitrite. We assessed S-nitrosylation and nitration of AJ-associated proteins to identify downstream NO redox signaling targets. We found that the GTPase-activating protein (GAP) p190RhoGAP-A was selectively nitrated at Tyr1105, resulting in impaired GAP activity and RhoA activation. Inhibition of eNOS or RhoA restored AJ integrity and diminished endothelial hyperpermeability in Cav-1(-/-) mice. Thrombin, a mediator of increased endothelial permeability, also induced nitration of p120-catenin-associated p190RhoGAP-A. Thus, eNOS-dependent nitration of p190RhoGAP-A represents a crucial mechanism for AJ disassembly and resultant increased endothelial permeability.     

Synergistic effects of ANP and sildenafil on cGMP levels and amelioration of acute hypoxic pulmonary hypertension

We hypothesized that the phosphodiesterase 5 inhibitor, sildenafil, and the guanosine cyclase stimulator, atrial natriuretic peptide (ANP), would act synergistically to increase cGMP levels and blunt hypoxic pulmonary hypertension in rats, because these compounds act via different mechanisms to increase the intracellular second messenger. Acute hypoxia: Adult Sprague-Dawley rats were gavaged with sildenafil (1 mg/ kg) or vehicle and exposed to acute hypoxia with and without ANP (10(-8)-10(-5) M ). Sildenafil decreased systemic blood pressure (103 +/- 10 vs. 87 +/- 6 mm Hg, P < 0.001) and blunted the hypoxia-induced increase in right ventricular systolic pressure (RVSP; percent increase 73.7% +/- 9.4% in sildenafil-treated rats vs. 117.2% +/- 21.1% in vehicle-treated rats, P = 0.03). Also, ANP and sildenafil had synergistic effects on blunting the hypoxia-induced increase in RVSP (P < 0.001) and on rising plasma cGMP levels (P < 0.05). Chronic hypoxia: Other rats were exposed to prolonged hypoxia (3 weeks, 0.5 atm) after subcutaneous implantation of a sustained-release pellet containing lower (2.5 mg), or higher (25 mg) doses of sildenafil, or placebo. Higher-dose, but not lower-dose sildenafil blunted the chronic hypoxia-induced increase in RVSP (P = 0.006). RVSP and plasma sildenafil levels were inversely correlated in hypoxic rats (r(2) = 0.68, P = 0.044). Lung cGMP levels were increased by both chronic hypoxia and sildenafil, with the greatest increase achieved by the combination. Plasma and right ventricular (RV) cGMP levels were increased by hypoxia, but sildenafil had no effect. RV hypertrophy and pulmonary artery muscularization were also unaffected by sildenafil. In conclusion, sildenafil and ANP have synergistic effects on the blunting of hypoxia-induced pulmonary vasoconstriction. During chronic hypoxia, sildenafil normalizes RVSP, but in the doses used, sildenafil has no effect on RV hypertrophy or pulmonary vascular remodeling.     

Divergent contractile and structural responses of the murine PKC-epsilon null pulmonary circulation to chronic hypoxia

Loss of PKC-epsilon limits the magnitude of acute hypoxic pulmonary vasoconstriction (HPV) in the mouse. Therefore, we hypothesized that loss of PKC-epsilon would decrease the contractile and/or structural response of the murine pulmonary circulation to chronic hypoxia (Hx). However, the pattern of lung vascular responses to chronic Hx may or may not be predicted by the acute HPV response. Adult PKC-epsilon wild-type (PKC-epsilon(+/+)), heterozygous null, and homozygous null (PKC-epsilon(-/-)) mice were exposed to normoxia or Hx for 5 wk. PKC-epsilon(-/-) mice actually had a greater increase in right ventricular (RV) systolic pressure, RV mass, and hematocrit in response to chronic Hx than PKC-epsilon(+/+) mice. In contrast to the augmented PA pressure and RV hypertrophy, pulmonary vascular remodeling was increased less than expected (i.e., equal to PKC-epsilon(+/+) mice) in both the proximal and distal PKC-epsilon(-/-) pulmonary vasculature. The contribution of increased vascular tone to this pulmonary hypertension (PHTN) was assessed by measuring the acute vasodilator response to nitric oxide (NO). Acute inhalation of NO reversed the increased PA pressure in hypoxic PKC-epsilon(-/-) mice, implying that the exaggerated PHTN may be due to a relative deficiency in nitric oxide synthase (NOS). Despite the higher PA pressure, chronic Hx stimulated less of an increase in lung endothelial (e) and inducible (i) NOS expression in PKC-epsilon(-/-) than PKC-epsilon(+/+) mice. In contrast, expression of nNOS in PKC-epsilon(+/+) mice decreased in response to chronic Hx, while lung levels in PKC-epsilon(-/-) mice remained unchanged. In summary, loss of PKC-epsilon results in increased vascular tone, but not pulmonary vascular remodeling in response to chronic Hx. Blunting of Hx-induced eNOS and iNOS expression may contribute to the increased vascular tone. PKC-epsilon appears to be an important signaling intermediate in the hypoxic regulation of each NOS isoform.     

Attenuation of acute hypoxic pulmonary vasoconstriction and hypoxic pulmonary hypertension in mice by inhibition of Rho-kinase

RhoA GTPase mediates a variety of cellular responses, including activation of the contractile apparatus, growth, and gene expression. Acute hypoxia activates RhoA and, in turn, its downstream effector, Rho-kinase, and previous studies in rats have suggested a role for Rho/Rho-kinase signaling in both acute and chronically hypoxic pulmonary vasoconstriction. We therefore hypothesized that activation of Rho/Rho-kinase in the pulmonary circulation of mice contributes to acute hypoxic pulmonary vasoconstriction and chronic hypoxia-induced pulmonary hypertension and vascular remodeling. In isolated, salt solution-perfused mouse lungs, acute administration of the Rho-kinase inhibitor Y-27632 (1 x 10(-5) M) attenuated hypoxic vasoconstriction as well as that due to angiotensin II and KCl. Chronic treatment with Y-27632 (30 mg x kg(-1) x day(-1)) via subcutaneous osmotic pump decreased right ventricular systolic pressure, right ventricular hypertrophy, and neomuscularization of the distal pulmonary vasculature in mice exposed to hypobaric hypoxia for 14 days. Analysis of a small number of proximal pulmonary arteries suggested that Y-27632 treatment reduced the level of phospho-CPI-17, a Rho-kinase target, in hypoxic lungs. We also found that endothelial nitric oxide synthase protein in hypoxic lungs was augmented by Y-27632, suggesting that enhanced nitric oxide production might have played a role in the Y-27632-induced attenuation of chronically hypoxic pulmonary hypertension. In conclusion, Rho/Rho-kinase activation is important in the effects of both acute and chronic hypoxia on the pulmonary circulation of mice, possibly by contributing to both vasoconstriction and vascular remodeling.     

PDE5A inhibition attenuates bleomycin-induced pulmonary fibrosis and pulmonary hypertension through inhibition of ROS generation and RhoA/Rho kinase activation

Pulmonary hypertension frequently complicates interstitial lung disease, where it is associated with a high mortality. Patients with this dual diagnosis often fare worse than those with pulmonary arterial hypertension (PAH) alone and respond poorly to standard PAH therapy, often dying of right ventricular (RV) failure. We hypothesize that nitric oxide synthase (NOS) uncoupling is important in the pathogenesis of interstitial lung disease-associated pulmonary hypertension, and this process can be abrogated by phosphodiesterase type 5 (PDE5) inhibition to improve pulmonary vascular remodeling and right ventricular function. Intratracheal bleomycin (4 U/kg) or saline control was administered to C57/BL6 mice after anesthesia. After recovery, animals were fed a diet of sildenafil (100 mg.kg(-1).day(-1)) or vehicle for 2 wk when they underwent hemodynamic measurements, and tissues were harvested. Survival was reduced in animals treated with bleomycin compared with controls and was improved with sildenafil (100.0 vs. 73.7 vs. 84.2%, P < 0.05). RV/LV+S ratio was higher in bleomycin-alone mice with improvement in ratio when sildenafil was administered (33.00 +/- 0.01% vs. 20.98 +/- 0.01% P < 0.05). Histology showed less pulmonary vascular and RV fibrosis in the group cotreated with sildenafil. Bleomycin was associated with a marked increase in superoxide generation by DHE histological staining and luminol activity in both heart and lung. Treatment with sildenafil resulted in a concomitant reduction in superoxide levels in both heart and lung. These data demonstrate that PDE5 inhibition ameliorates RV hypertrophy and pulmonary fibrosis associated with intratracheal bleomycin in a manner that is associated with improved NOS coupling and a reduction in reactive oxygen species signaling.     

Bosentan inhibits oxidative and nitrosative stress and rescues occlusive pulmonaryhypertension

Pulmonary arterial hypertension (PH) is a fatal disease marked by excessive pulmonary vascular cell proliferation. Patients with idiopathic PH express endothelin-1 (ET-1) at high levels in their lungs. As the activation of both types of ET-1 receptor (ETA and ETB) leads to increased generation of superoxide and hydrogen peroxide, this may contribute to the severe oxidative stress found in PH patients. As a number of pathways may induce oxidative stress, the particular role of ET-1 remains unclear. The aim of this study was to determine whether inhibition of ET-1 signaling could reduce pulmonary oxidative stress and attenuate the progression of disease in rats with occlusive-angioproliferative PH induced by a single dose of SU5416 (200 mg/kg) and subsequent exposure to hypoxia for 21 days. Using this regimen, animals developed severe PH as evidenced by a progressive increase in right-ventricle (RV) peak systolic pressure (RVPSP), severe RV hypertrophy, and pulmonary endothelial and smooth muscle cell proliferation, resulting in plexiform vasculopathy. PH rats also had increased oxidative stress, correlating with endothelial nitric oxide synthase uncoupling and NADPH oxidase activation, leading to enhanced protein nitration and increases in markers of vascular remodeling. Treatment with the combined ET receptor antagonist bosentan (250 mg/kg/day; day 10 to 21) prevented further increase in RVPSP and RV hypertrophy, decreased ETA/ETB protein levels, reduced oxidative stress and protein nitration, and resulted in marked attenuation of pulmonary vascular cell proliferation. We conclude that inhibition of ET-1 signaling significantly attenuates the oxidative and nitrosative stress associated with PH and prevents its progression.     

Role of VEGF-B in the lung during development of chronic hypoxic pulmonary hypertension

Angiogenic factors exert protective effects on the lung. To investigate the effect of VEGF-B, a factor coexpressed in the lung with VEGF-A, we assessed chronic hypoxic pulmonary hypertension in VEGF-B knockout mice (VEGF-B-/-) and in rats with lung overexpression of VEGF-B induced by adenovirus transfer. No significant difference in pulmonary hemodynamics, right ventricular hypertrophy, distal vessel muscularization, or vascular density was found between VEGF-B-/- and control mice after 3 wk of hypoxia. When overexpressed, VEGF-B(167) or VEGF-B(186) had protective effects similar to those of human VEGF-A(165). Lung endothelial nitric oxide synthase (eNOS) expression was increased by 5 days of hypoxia or VEGF-A adenovirus vector (Ad.VEGF-A) overexpression, whereas VEGF-B(167) or VEGF-B(186) had no effect. With hypoxia or normoxia, the wet-to-dry lung weight ratio was increased 5 days after Ad.VEGF-A administration compared with control (Ad.nul), Ad.VEGF-B(167), or Ad.VEGF-B(186). Endogenous VEGF-B does not counteract the development of hypoxic pulmonary hypertension. However, when overexpressed in the lung, VEGF-B can be as potent as VEGF-A in attenuating pulmonary hypertension, although it has no effect on eNOS expression or vascular permeability.     

Nitrotyrosinylation,remodeling and endothelial‐myocyte uncoupling in iNOS,cystathionine beta synthase (CBS) knockouts and iNOS/CBS double knockout mice

Increased levels of homocysteine (Hcy), recognized as hyperhomocysteinemia (HHcy), were associated with cardiovascular diseases. There was controversy regarding the detrimental versus cardio protective role of inducible nitric oxide synthase (iNOS) in ischemic heart disease. The aim of this study was to test the hypothesis that the Hcy generated nitrotyrosine by inducing the endothelial nitric oxide synthase, causing endothelial‐myocyte (E‐M) coupling. To differentiate the role of iNOS versus constitutive nitric oxide synthase (eNOS and nNOS) in Hcy‐mediated nitrotyrosine generation and matrix remodeling in cardiac dysfunction, left ventricular (LV) tissue was analyzed from cystathionine beta synthase (CBS) heterozygote knockout, iNOS homozygote knockout, CBS?/+/iNOS?/? double knockout, and wild‐type (WT) mice. The levels of nitrotyrosine, MMP‐2 and ‐9 (zymographic analysis), and fibrosis (by trichrome stain) were measured. The endothelial‐myocyte function was determined in cardiac rings. In CBS?/+ mice, homocysteine was elevated and in iNOS?/? mice, nitric oxide was significantly reduced. The nitrotyrosine and matrix metalloproteinase‐9 (MMP‐9) levels were elevated in double knockout and CBS?/+ as compared to WT mice. Although MMP‐2 levels were similar in CBS?/+, iNOS?/?, and CBS?/+/iNOS?/?, the levels were three‐ to fourfold higher than WT. The levels of collagen were similar in CBS?/+ and iNOS?/?, but they were threefold higher than WT. Interesting, the levels of collagen increased sixfold in double knockouts, compared to WT, suggesting synergism between high Hcy and lack of iNOS. Left ventricular hypertrophy was exaggerated in the iNOS?/? and double knockout, and mildly increased in the CBS?/+, compared to WT mice. The endothelial‐dependent relaxation was attenuated to the same extent in the CBS?/+ and iNOS?/?, compared to WT, but it was robustly blunted in double knockouts. The results concluded that homocysteine generated nitrotyrosine in the vicinity of endothelium, caused MMP activation and endothelium‐myocyte uncoupling. The generation of nitrotyrosine was independent of iNOS. J. Cell. Biochem. 106: 119–126, 2009. © 2008 Wiley‐Liss, Inc.     

Upregulation of nitric oxide synthase in mice with severe hypoxia-induced pulmonary hypertension

Background  

The importance of nitric oxide (NO) in hypoxic pulmonary hypertension has been demonstrated using nitric oxide synthase (NOS) knockout mice. In that model NO from endothelial NOS (eNOS) plays a central role in modulating pulmonary vascular tone and attenuating hypoxic pulmonary hypertension. However, the normal regulation of NOS expression in mice following hypoxia is uncertain. Because genetically engineered mice are often utilized in studies of NO, we conducted the present study to determine how hypoxia alters NOS expression in wild-type mice.     

Pulmonary hypertension in the newborn GTP cyclohydrolase I-deficient mouse

Tetrahydrobiopterin (BH4) is a regulator of endothelial nitric oxide synthase (eNOS) activity. Deficient levels result in eNOS uncoupling, with a shift from nitric oxide to superoxide generation. The hph-1 mutant mouse has deficient GTP cyclohydrolase I (GTPCH1) activity, resulting in low BH4 tissue content. The adult hph-1 mouse has pulmonary hypertension, but whether such condition is present from birth is not known. Thus, we evaluated newborn animals’ pulmonary arterial medial thickness, biopterin content (BH4 + BH2), H₂O₂ and eNOS, right ventricle-to-left ventricle + septum (RV/LV + septum) ratio, near-resistance pulmonary artery agonist-induced force, and endothelium-dependent and -independent relaxation. The lung biopterin content was inversely related to age for both types, but significantly lower in hph-1 mice, compared to wild-type animals. As judged by the RV/LV + septum ratio, newborn hph-1 mice have pulmonary hypertension and, after a 2-week 13% oxygen exposure, the ratios were similar in both types. The pulmonary arterial agonist-induced force was reduced (P < 0.01) in hph-1 animals and no type-dependent difference in endothelium-dependent or -independent vasorelaxation was observed. Compared to wild-type mice, the lung H₂O₂ content was increased, whereas the eNOS expression was decreased (P < 0.01) in hph-1 animals. The pulmonary arterial medial thickness, a surrogate marker of vascular remodeling, was increased (P < 0.01) in hph-1 compared to wild-type mice. In conclusion, our data suggest that pulmonary hypertension is present from birth in the GTPCH1-deficient mice, not as a result of impaired vasodilation, but secondary to vascular remodeling.     

Mice deficient in galectin-1 exhibit attenuated physiological responses to chronic hypoxia-induced pulmonary hypertension

Pulmonary hypertension (PH) is characterized by sustained vasoconstriction, with subsequent extracellular matrix (ECM) production and smooth muscle cell (SMC) proliferation. Changes in the ECM can modulate vasoreactivity and SMC contraction. Galectin-1 (Gal-1) is a hypoxia-inducible beta-galactoside-binding lectin produced by vascular, interstitial, epithelial, and immune cells. Gal-1 regulates SMC differentiation, proliferation, and apoptosis via interactions with the ECM, as well as immune system function, and, therefore, likely plays a role in the pathogenesis of PH. We investigated the effects of Gal-1 during hypoxic PH by quantifying 1) Gal-1 expression in response to hypoxia in vitro and in vivo and 2) the effect of Gal-1 gene deletion on the magnitude of the PH response to chronic hypoxia in vivo. By constructing and screening a subtractive library, we found that acute hypoxia increases expression of Gal-1 mRNA in isolated pulmonary mesenchymal cells. In wild-type (WT) mice, Gal-1 immunoreactivity increased after 6 wk of hypoxia. Increased expression of Gal-1 protein was confirmed by quantitative Western analysis. Gal-1 knockout (Gal-1(-/-)) mice showed a decreased PH response, as measured by right ventricular pressure and the ratio of right ventricular to left ventricular + septum wet weight compared with their WT counterparts. However, the number and degree of muscularized vessels increased similarly in WT and Gal-1(-/-) mice. In response to chronic hypoxia, the decrease in factor 8-positive microvessel density was similar in both groups. Vasoreactivity of WT and Gal-1(-/-) mice was tested in vivo and with use of isolated perfused lungs exposed to acute hypoxia. Acute hypoxia caused a significant increase in RV pressure in wild-type and Gal-1(-/-) mice; however, the response of the Gal-1(-/-) mice was greater. These results suggest that Gal-1 influences the contractile response to hypoxia and subsequent remodeling during hypoxia-induced PH, which influences disease progression.     

Endothelial nitric oxide synthase (NOS3) knockout decreases NOS2 induction, limiting hyperoxygenation and conferring protection in the postischemic heart

Although it has been shown that endothelial nitric oxide synthase (eNOS)-derived nitric oxide downregulates mitochondrial oxygen consumption during early reperfusion, its effects on inducible NOS (iNOS) induction and myocardial injury during late reperfusion are unknown. Wild-type (WT) and eNOS(-/-) mice were subjected to 30 min of coronary ligation followed by reperfusion. Expression of iNOS mRNA and protein levels and peroxynitrite production were lower in postischemic myocardium of eNOS(-/-) mice than levels in WT mice 48 h postreperfusion. Significantly improved hemodynamics (+/-dP/dt, left ventricular systolic pressure, mean arterial pressure), increased rate pressure product, and reduced myocardial infarct size (18 +/- 2.5% vs. 31 +/- 4.6%) were found 48 h after reperfusion in eNOS(-/-) mice compared with WT mice. Myocardial infarct size was also significantly decreased in WT mice treated with the specific iNOS inhibitor 1400W (20.5 +/- 3.4%) compared with WT mice treated with PBS (33.9 +/- 5.3%). A marked reperfusion-induced hyperoxygenation state was observed by electron paramagnetic resonance oximetry in postischemic myocardium, but Po(2) values were significantly lower from 1 to 72 h in eNOS(-/-) than in WT mice. Cytochrome c-oxidase activity and NADH dehydrogenase activity were significantly decreased in postischemic myocardium in WT and eNOS(-/-) mice compared with baseline control, respectively, and NADH dehydrogenase activity was significantly higher in eNOS(-/-) than in WT mice. Thus deficiency of eNOS exerted a sustained beneficial effect on postischemic myocardium 48 h after reperfusion with preserved mitochondrial function, which appears to be due to decreased iNOS induction and decreased iNOS-derived peroxynitrite in postischemic myocardium.     

Berberine attenuates hypoxia-induced pulmonary arterial hypertension via bone morphogenetic protein and transforming growth factor-β signaling

Hypoxia-induced excessive pulmonary artery smooth muscle cell (PASMC) proliferation plays an important role in the pathology of pulmonary arterial hypertension (PAH). Berberine (BBR) is reported as an effective antiproliferative properties applied in clinical. However, the effect of BBR on PAH remains unclear. In the present study, we elucidated the protective effects of BBR against abnormal PASMC proliferation and vascular remodeling in chronic hypoxia-induced hearts. Furthermore, the potential mechanisms of BBR were investigated. For this purpose, C57/BL6 mice were exposed to chronic hypoxia for 4 weeks to mimic severe PAH. Hemodynamic and pulmonary pathomorphology data showed that chronic hypoxia significantly increased the right ventricular systolic pressure (RVSP), the right ventricle/left ventricle plus septum RV/(LV + S) weight ratio, and the median width of pulmonary arterioles. BBR attenuated the elevations in RVSP and RV/(LV + S) and mitigated pulmonary vascular structure remodeling. BBR also suppressed the hypoxia-induced increases in the expression of proliferating cell nuclear antigen (PCNA) and of α-smooth muscle actin. Furthermore, administration of BBR significantly increased the expression of bone morphogenetic protein type II receptor (BMPR-II) and its downstream molecules P-smad1/5 and decreased the expression of transforming growth factor-β (TGF-β) and its downstream molecules P-smad2/3. Moreover, peroxisome proliferator-activated receptor γ expression was significantly decreased in the hypoxia group, and this decrease was reversed by BBR treatment. Our study demonstrated that the protective effect of BBR against hypoxia-induced PAH in a mouse model may be achieved through altered BMPR-II and TGF-β signaling.     

Arterial remodeling and plasma volume expansion in caveolin-1-deficient mice

Caveolin-1 (Cav-1) is essential for the morphology of membrane caveolae and exerts a negative influence on a number of signaling systems, including nitric oxide (NO) production and activity of the MAP kinase cascade. In the vascular system, ablation of caveolin-1 may thus be expected to cause arterial dilatation and increased vessel wall mass (remodeling). This was tested in Cav-1 knockout (KO) mice by a detailed morphometric and functional analysis of mesenteric resistance arteries, shown to lack caveolae. Quantitative morphometry revealed increased media thickness and media-to-lumen ratio in KO. Pressure-induced myogenic tone and flow-induced dilatation were decreased in KO arteries, but both were increased toward wild-type (WT) levels following NO synthase (NOS) inhibition. Isometric force recordings following NOS inhibition showed rightward shifts of passive and active length-force relationships in KO, and the force response to alpha(1)-adrenergic stimulation was increased. In contrast, media thickness and force response of the aorta were unaltered in KO vs. WT, whereas lumen diameter was increased. Mean arterial blood pressure during isoflurane anesthesia was not different in KO vs. WT, but greater fluctuation in blood pressure over time was noted. Following NOS inhibition, fluctuations disappeared and pressure increased twice as much in KO (38 +/- 6%) compared with WT (17 +/- 3%). Tracer-dilution experiments showed increased plasma volume in KO. We conclude that NO affects blood pressure more in Cav-1 KO than in WT mice and that restructuring of resistance vessels and an increased responsiveness to adrenergic stimulation compensate for a decreased tone in Cav-1 KO mice.