Zhongjie Sun

Klotho Gene Delivery Prevents the Progression of Spontaneous Hypertension and Renal Damage

Klotho is a recently discovered antiaging gene. The objective of this study was to test the hypothesis that klotho gene delivery attenuates the progression of spontaneous hypertension and renal damage in spontaneous hypertensive rats (SHRs). An adeno-associated virus (AAV) carrying mouse klotho full-length cDNA (AAV.mKL) was constructed for in vivo expression of klotho. Four groups of male SHRs and 1 group of sex- and age-matched Wistar-Kyoto rats (5 rats per group) were used. Blood pressure was measured twice in all of the animals before gene delivery. Four groups of SHRs received an IV injection of AAV.mKL, AAV.LacZ, AAV.GFP, and PBS, respectively. The Wistar-Kyoto group received PBS and served as a control. AAV.mKL stopped the further increase in blood pressure in SHRs, whereas blood pressures continued to increase in other SHR groups. One single dose of AAV.mKL prevented the progression of spontaneous hypertension for at least 12 weeks (length of the study). Klotho expression and production were suppressed in SHRs, which were reverted by AAV.mKL. AAV.mKL increased plasma interleukin 10 levels but decreased Nox2 expression, NADPH oxidase activity, and superoxide production in kidneys and aortas in SHRs. AAV.mKL abolished renal tubular atrophy and dilation, tubular deposition of proteinaceous material, glomerular collapse, and collagen deposition seen in SHRs, indicating that klotho gene delivery attenuated renal damage. Therefore, the suppressed klotho expression may play a role in the progression of spontaneous hypertension and renal damage in SHRs. AAV delivery of klotho may offer a new approach for the long-term control of hypertension and for renoprotection.

Aging, Arterial Stiffness, and Hypertension

Although the etiology of essential hypertension remains unknown, it is clear that multiple factors may contribute to the pathogenesis of hypertension. Hypertension is an outcome of the interaction of multiple genetic and environmental factors. Several epidemiological studies indicated that the incidence of arterial stiffness and hypertension and related cardiovascular disease (stroke, myocardial infarction) is higher in the aged than in the young population.1–4 The prevalence of arterial stiffening and hypertension increases with age.1,5 Based on an epidemiological study,6 the prevalence of hypertension is more than doubled in the elderly than in the young population. More than two-thirds of individuals after 65 years of age experience hypertension according to the Seventh Report of the Joint National Committee (JNC-7).7 Therefore, it is generally thought that hypertension is an aging disorder. In recent years, metabolic syndrome and hypertension are increasingly seen in the middle-aged and young populations. In these subpopulations, insulin resistance and overproduction of adipokines impair endothelial and heart function leading to early and accelerated cardiovascular aging. It was reported that premature aging (progeria) is associated with accelerated vascular stiffening or vascular aging.8nnAging is defined as the age-related decline in physiological function essential for survival and fertility. Cardiovascular aging is an important factor that determines life span. The wall of large conduit arteries, especially aorta, thicken and lose elasticity over time, and this process results in an increase in pulse wave velocity, an important and reliable measure of arterial stiffness. The increased arterial stiffness, whatever its underlying causes, would reduce the reservoir/buffering function of the conduit arteries near the heart and increase pulse wave velocity, both of which increase systolic and pulse pressure. Therefore, aging-related hypertension is characterized by a significant increase in systolic blood pressure with no change or even a …

Nitric oxide, oxidative stress and inflammation in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a chronic and progressive disease characterized by a persistent elevation of pulmonary artery pressure accompanied by right ventricular hypertrophy (RVH). The current treatment for pulmonary hypertension is limited and only provides symptomatic relief due to unknown cause and pathogenesis of the disease. Both vasoconstriction and structural remodeling (enhanced proliferation of vascular smooth muscle cell) of the pulmonary arteries contribute to the progressive course of PAH, irrespective of different underlying causes. The exact molecular mechanism of PAH, however, is not fully understood. The purpose of this review is to provide recent advances in the mechanistic investigation of PAH. Specifically, this review focuses on nitric oxide, oxidative stress and inflammation and how these factors contribute to the development and progression of PAH. This review also discusses recent and potential therapeutic advancements for the treatment of PAH.

Molecular Mechanisms of Pulmonary Arterial Remodeling

Pulmonary arterial hypertension (PAH) is characterized by a persistent elevation of pulmonary arterial pressure and pulmonary arterial remodeling with unknown etiology. Current therapeutics available for PAH are primarily directed at reducing the pulmonary blood pressure through their effects on the endothelium. It is well accepted that pulmonary arterial remodeling is primarily due to excessive pulmonary arterial smooth muscle cell (PASMC) proliferation that leads to narrowing or occlusion of the pulmonary vessels. Future effective therapeutics will be successful in reversing the vascular remodeling in the pulmonary arteries and arterioles. The purpose of this review is to provide updated information on molecular mechanisms involved in pulmonary arterial remodeling with a focus on growth factors, transcription factors, and epigenetic pathways in PASMC proliferation. In addition, this review will highlight novel therapeutic strategies for potentially reversing PASMC proliferation.

Ribonucleic Acid Interference Knockdown of Interleukin 6 Attenuates Cold-Induced Hypertension

The purpose of this study was to determine the role of the proinflammatory cytokine interleukin (IL) 6 in cold-induced hypertension. Four groups of male Sprague-Dawley rats were used (6 rats per group). After blood pressure was stabilized, 3 groups received intravenous delivery of adenoassociated virus carrying IL-6 small hairpin RNA (shRNA), adenoassociated virus carrying scrambled shRNA, and PBS, respectively, before exposure to a cold environment (5°C). The last group received PBS and was kept at room temperature (25°C, warm) as a control. Adenoassociated virus delivery of IL-6 shRNA significantly attenuated cold-induced elevation of systolic blood pressure and kept it at the control level for ≤7 weeks (length of the study). Chronic exposure to cold upregulated IL-6 expression in aorta, heart, and kidneys and increased macrophage and T-cell infiltration in kidneys, suggesting that cold exposure increases inflammation. IL-6 shRNA delivery abolished the cold-induced upregulation of IL-6, indicating effective silence of IL-6. Interestingly, RNA interference knockdown of IL-6 prevented cold-induced inflammation, as evidenced by a complete inhibition of tumor necrosis factor-&agr; expression and leukocyte infiltration by IL-6 shRNA. RNA interference knockdown of IL-6 significantly decreased the cold-induced increase in vascular superoxide production. It is noted that IL-6 shRNA abolished the cold-induced increase in collagen deposition in the heart, suggesting that inflammation is involved in cold-induced cardiac remodeling. Cold exposure caused glomerular collapses, which could be prevented by knockdown of IL-6, suggesting an important role of inflammation in cold-induced renal damage. In conclusion, cold exposure increased IL-6 expression and inflammation, which play critical roles in the pathogenesis of cold-induced hypertension and cardiac and renal damage.

Antiaging Gene Klotho Deficiency Promoted High-Fat Diet–Induced Arterial Stiffening via Inactivation of AMP-Activated Protein Kinase

Klotho was originally discovered as an aging-suppressor gene. The objective of this study is to investigate whether klotho gene deficiency affects high-fat diet (HFD)–induced arterial stiffening. Heterozygous Klotho-deficient (KL+/−) mice and WT littermates were fed on HFD or normal diet. HFD increased pulse wave velocity within 5 weeks in KL+/− mice but not in wild-type mice, indicating that klotho deficiency accelerates and exacerbates HFD-induced arterial stiffening. A greater increase in blood pressure was found in KL+/− mice fed on HFD. Protein expressions of phosphorylated AMP-activated protein kinase-&agr; (AMPK&agr;), phosphorylated endothelial nitric oxide synthase (eNOS), and manganese-dependent superoxide dismutase (Mn-SOD) were decreased, whereas protein expressions of collagen I, transforming growth factor-&bgr;1, and Runx2 were increased in aortas of KL+/− mice fed on HFD. Interestingly, daily injections of an AMPK&agr; activator, 5-aminoimidazole-4-carboxamide-3-ribonucleoside, abolished the increases in pulse wave velocity, blood pressure, and blood glucose in KL+/− mice fed on HFD. Treatment with 5-aminoimidazole-4-carboxamide-3-ribonucleoside for 2 weeks not only abolished the downregulation of phosphorylated AMPK&agr;, phosphorylated eNOS, and Mn-SOD levels but also attenuated the increased levels of collagen I, transforming growth factor-&bgr;1, Runx2, superoxide, elastic lamellae breaks, and calcification in aortas of KL+/− mice fed on HFD. In cultured mouse aortic smooth muscle cells, cholesterol plus KL-deficient serum decreased phosphorylation levels of AMPK&agr; and LKB1 (an important upstream regulator of AMPK&agr; activity) but increased collagen I synthesis, which can be eliminated by activation of AMPK&agr; by 5-aminoimidazole-4-carboxamide-3-ribonucleoside. In conclusions, Klotho deficiency promoted HFD-induced arterial stiffening and hypertension via downregulation of AMPK&agr; activity.

Inhibition of Phosphodiesterase-1 Attenuates Cold-Induced Pulmonary Hypertension

Chronic exposure to cold caused pulmonary arterial hypertension (cold-induced pulmonary hypertension [CIPH]) and increased phosphodiesterase-1C (PDE-1C) expression in pulmonary arteries (PAs) in rats. The purpose of this study is to investigate a hypothesis that inhibition of PDE-1 would decrease inflammatory infiltrates and superoxide production leading to attenuation of CIPH. Three groups of male rats were exposed to moderate cold (5±1°C) continuously, whereas 3 groups were maintained at room temperature (23.5±1°C, warm; 6 rats/group). After 8-week exposure to cold, 3 groups in each temperature condition received continuous intravenous infusion of 8-isobutyl-methylxanthine (8-IBMX) (PDE-1 inhibitor), apocynin (NADPH oxidase inhibitor) or vehicle, respectively, for 1 week. Cold exposure significantly increased right-ventricular systolic pressure compared with warm groups (33.8±3.2 versus 18.6±0.3 mm Hg), indicating that animals developed CIPH. Notably, treatment with 8-IBMX significantly attenuated the cold-induced increase in right ventricular pressure (23.5±1.8 mm Hg). Cold exposure also caused right-ventricular hypertrophy, whereas 8-IBMX reversed cold-induced right ventricular hypertrophy. Cold exposure increased PDE-1C protein expression, macrophage infiltration, NADPH oxidase activity, and superoxide production in PAs and resulted in PA remodeling. 8-IBMX abolished cold-induced upregulation of PDE-1C in PAs. Interestingly, inhibition of PDE-1 eliminated cold-induced macrophage infiltration, NADPH oxidase activation, and superoxide production in PAs and reversed PA remodeling. Inhibition of NADPH oxidase by apocynin abolished cold-induced superoxide production and attenuated CIPH and PA remodeling. In conclusion, inhibition of PDE-1 attenuated CIPH and reversed cold-induced PA remodeling by suppressing macrophage infiltration and superoxide production, suggesting that upregulation of PDE-1C expression may be involved in the pathogenesis of CIPH.

Activation of SIRT1 Attenuates Klotho Deficiency-Induced Arterial Stiffness and Hypertension by Enhancing AMP-Activated Protein Kinase Activity.

Arterial stiffness is an independent risk factor for stroke and myocardial infarction. This study was designed to investigate the role of SIRT1, an important deacetylase, and its relationship with Klotho, a kidney-derived aging-suppressor protein, in the pathogenesis of arterial stiffness and hypertension. We found that the serum level of Klotho was decreased by ≈45% in patients with arterial stiffness and hypertension. Interestingly, Klotho haplodeficiency caused arterial stiffening and hypertension, as evidenced by significant increases in pulse wave velocity and blood pressure in Klotho-haplodeficient (KL +/– ) mice. Notably, the expression and activity of SIRT1 were decreased significantly in aortic endothelial and smooth muscle cells in KL +/– mice, suggesting that Klotho deficiency downregulates SIRT1. Treatment with SRT1720 (15 mg/kg/d, IP), a specific SIRT1 activator, abolished Klotho deficiency–induced arterial stiffness and hypertension in KL +/– mice. Klotho deficiency was associated with significant decreases in activities of AMP-activated protein kinase &agr; (AMPK&agr;) and endothelial NO synthase (eNOS) in aortas, which were abolished by SRT1720. Furthermore, Klotho deficiency upregulated NADPH oxidase activity and superoxide production, increased collagen expression, and enhanced elastin fragmentation in the media of aortas. These Klotho deficiency–associated changes were blocked by SRT1720. In conclusion, this study provides the first evidence that Klotho deficiency downregulates SIRT1 activity in arterial endothelial and smooth muscle cells. Pharmacological activation of SIRT1 may be an effective therapeutic strategy for arterial stiffness and hypertension.

AAV Delivery of Tumor Necrosis Factor-α Short Hairpin RNA Attenuates Cold-Induced Pulmonary Hypertension and Pulmonary Arterial Remodeling

Cold temperatures are associated with increased mortality and morbidity of cardiovascular and pulmonary disease. Cold exposure causes lung inflammation, pulmonary hypertension (PH), and right ventricle hypertrophy, but there is no effective therapy because of unknown mechanism. Here, we investigated whether RNA interference silencing of tumor necrosis factor (TNF)-&agr; decreases cold-induced macrophage infiltration, PH, and pulmonary arterial (PA) remodeling. We found for the first time that continuous cold exposure (5.0°C) increased TNF-&agr; expression and macrophage infiltration in the lungs and PAs right before elevation of right ventricle systolic pressure. The in vivo RNA interference silencing of TNF-&agr; was achieved by intravenous delivery of recombinant AAV-2 carrying TNF-&agr; short hairpin small–interfering RNA 24 hours before cold exposure. Cold exposure for 8 weeks significantly increased right ventricle pressure compared with the warm controls (40.19±4.9 versus 22.9±1.1 mm Hg), indicating that cold exposure caused PH. Cold exposure increased TNF-&agr;, interleukin-6, and phosphodiesterase-1C protein expression in the lungs and PAs and increased lung macrophage infiltration. Notably, TNF-&agr; short hairpin small–interfering RNA prevented the cold-induced increases in TNF-&agr;, interleukin-6, and phosphodiesterase-1C protein expression, abolished lung macrophage infiltration, and attenuated PH (26.28±1.6 mm Hg), PA remodeling, and right ventricle hypertrophy. PA smooth muscle cells isolated from cold-exposed animals showed increased intracellular superoxide levels and cell proliferation along with decreased intracellular cGMP. These cold-induced changes were prevented by TNF-&agr; short hairpin small–interfering RNA. In conclusions, upregulation of TNF-&agr; played a critical role in the pathogenesis of cold-induced PH by promoting pulmonary macrophage infiltration and inflammation. AAV delivery of TNF-&agr; short hairpin small–interfering RNA may be an effective therapeutic approach for cold-induced PH and PA remodeling.

Normal IgG Downregulates the Intracellular Superoxide Level and Attenuates Migration and Permeability in Human Aortic Endothelial Cells Isolated From a Hypertensive Patient

The normal IgG, a circulating antibody, is maintained at a constant level in humans. However, little is known regarding whether normal IgG has effects on the function of vascular endothelial cells. The purpose of this study was to investigate whether IgG affects superoxide (O2 ·−) generation and cell permeability in human aortic endothelial cells (HAECs) isolated from a hypertensive patient. The effect of normal human IgG on endothelial cell function was investigated in cultured HAECs isolated from a hypertensive patient who died of stroke. The results demonstrated, for the first time, that normal IgG attenuated the intracellular O2 ·− level and decreased cell migration, cell permeability, and stress fiber formation in HAECs. IgG significantly decreased Rac1 activity and NADPH oxidase activity but upregulated Mn superoxide dismutase expression in HAECs, which may contribute to the IgG-induced decrease in O2 ·− level. It is noted that AMP-activated protein kinase (AMPK) was activated by IgG, as evidenced by increased phosphorylation of AMPK. Interestingly, inhibition of AMPK by an AMPK inhibitor abolished IgG-induced decreases in Rac1 and NADPH oxidase activities and IgG-induced increases in Mn superoxide dismutase expression, suggesting that AMPK is an important mediator of the IgG-induced regulation of these enzymes. Importantly, inhibition of AMPK activity also prevented the IgG-induced decrease in O2 ·− levels, cell migration, cell permeability, and stress fiber formation. Therefore, normal human IgG may protect HAECs via activation of AMPK and subsequent decreases in intracellular O2 ·−. These findings reveal a previously unidentified role of normal IgG in regulating AMPK and endothelial cell function.