Endothelial Sp1/Sp3 are essential to the effect of captopril on blood pressure in male mice.

Endothelial dysfunction represents a major cardiovascular risk factor for hypertension. Sp1 and Sp3 belong to the specificity protein and Krüppel-like transcription factor families. They are ubiquitously expressed and closely associated with cardiovascular development. We investigate the role of Sp1 and Sp3 in endothelial cells in vivo and evaluate whether captopril, an angiotensin-converting enzyme inhibitor (ACEI), targets Sp1/Sp3 to exert its effects. Inducible endothelial-specific Sp1/Sp3 knockout mice are generated to elucidate their role in endothelial cells. Tamoxifen-induced deletion of endothelial Sp1 and Sp3 in male mice decreases the serum nitrite/nitrate level, impairs endothelium-dependent vasodilation, and causes hypertension and cardiac remodeling. The beneficial actions of captopril are abolished by endothelial-specific deletion of Sp1/Sp3, indicating that they may be targets for ACEIs. Captopril increases Sp1/Sp3 protein levels by recruiting histone deacetylase 1, which elevates deacetylation and suppressed degradation of Sp1/Sp3. Sp1/Sp3 represents innovative therapeutic target for captopril to prevent cardiovascular diseases.

Angiotensin-converting enzyme inhibitor promotes angiogenesis through Sp1/Sp3-mediated inhibition of notch signaling in male mice.

Angiogenesis is a critical pathophysiological process involved in organ growth and various diseases. Transcription factors Sp1/Sp3 are necessary for fetal development and tumor growth. Sp1/Sp3 proteins were downregulated in the capillaries of the gastrocnemius in patients with critical limb ischemia samples. Endothelial-specific Sp1/Sp3 knockout reduces angiogenesis in retinal, pathological, and tumor models and induced activation of the Notch1 pathway. Further, the inactivation of VEGFR2 signaling by Notch1 contributes to the delayed angiogenesis phenotype. Mechanistically, endothelial Sp1 binds to the promoter of Notch1 and inhibits its transcription, which is enhanced by Sp3. The proangiogenic effect of ACEI is abolished in Sp1/Sp3-deletion male mice. We identify USP7 as an ACEI-activated deubiquitinating enzyme that translocated into the nucleus binding to Sp1/Sp3, which are deacetylated by HDAC1. Our findings demonstrate a central role for endothelial USP7-Sp1/Sp3-Notch1 signaling in pathophysiological angiogenesis in response to ACEI treatment.

Smooth muscle-specific HuR knockout induces defective autophagy and atherosclerosis.

Human antigen R (HuR) is a widespread RNA-binding protein involved in homeostatic regulation and pathological processes in many diseases. Atherosclerosis is the leading cause of cardiovascular disease and acute cardiovascular events. However, the role of HuR in atherosclerosis remains unknown. In this study, mice with smooth muscle-specific HuR knockout (HuRSMKO) were generated to investigate the role of HuR in atherosclerosis. HuR expression was reduced in atherosclerotic plaques. As compared with controls, HuRSMKO mice showed increased plaque burden in the atherosclerotic model. Mechanically, HuR could bind to the mRNAs of adenosine 5'-monophosphate-activated protein kinase (AMPK) α1 and AMPKα2, thus increasing their stability and translation. HuR deficiency reduced p-AMPK and LC3II levels and increased p62 level, thereby resulting in defective autophagy. Finally, pharmacological AMPK activation induced autophagy and suppressed atherosclerosis in HuRSMKO mice. Our findings suggest that smooth muscle HuR has a protective effect against atherosclerosis by increasing AMPK-mediated autophagy.

LKB1IP promotes pathological cardiac hypertrophy by targeting PTEN/Akt signalling pathway.

Pathological cardiac hypertrophy represents a leading cause of morbidity and mortality worldwide. Liver kinase B1 interacting protein 1 (LKB1IP) was identified as the binding protein of tumour suppressor LKB1. However, the role of LKB1IP in the development of pathological cardiac hypertrophy has not been explored. The aim of this study was to investigate the function of LKB1IP in cardiac hypertrophy in response to hypertrophic stimuli. We investigated the cardiac level of LKB1IP in samples from patients with heart failure and mice with cardiac hypertrophy induced by isoproterenol (ISO) or transverse aortic constriction (TAC). LKB1IP knockout mice were generated and challenged with ISO injection or TAC surgery. Cardiac function, hypertrophy and fibrosis were then examined. LKB1IP expression was significantly up-regulated on hypertrophic stimuli in both human and mouse cardiac samples. LKB1IP knockout markedly protected mouse hearts against ISO- or TAC-induced cardiac hypertrophy and fibrosis. LKB1IP overexpression aggravated ISO-induced cardiomyocyte hypertrophy, and its inhibition attenuated hypertrophy in vitro. Mechanistically, LKB1IP activated Akt signalling by directly targeting PTEN and then inhibiting its phosphatase activity. In conclusion, LKB1IP may be a potential target for pathological cardiac hypertrophy.

HuR (Human Antigen R) Regulates the Contraction of Vascular Smooth Muscle and Maintains Blood Pressure.

OBJECTIVE: HuR (human antigen R)-an RNA-binding protein-is involved in regulating mRNA stability by binding adenylate-uridylate-rich elements. This study explores the role of HuR in the regulation of smooth muscle contraction and blood pressure. Approach and Results: Vascular HuRSMKO (smooth muscle-specific HuR knockout) mice were generated by crossbreeding HuRflox/flox mice with α-SMA (α-smooth muscle actin)-Cre mice. As compared with CTR (control) mice, HuRSMKO mice showed hypertension and cardiac hypertrophy. HuR levels were decreased in aortas from hypertensive patients and SHRs (spontaneously hypertensive rats), and overexpression of HuR could lower the blood pressure of SHRs. Contractile response to vasoconstrictors was increased in mesenteric artery segments isolated from HuRSMKO mice. The functional abnormalities in HuRSMKO mice were attributed to decreased mRNA and protein levels of RGS (regulator of G-protein signaling) protein(s) RGS2, RGS4, and RGS5, which resulted in increased intracellular calcium increase. Consistently, the degree of intracellular calcium ion increase in HuR-deficient smooth muscle cells was reduced by overexpression of RGS2, RGS4, or RGS5. Finally, administration of RGS2 and RGS5 reversed the elevated blood pressure in HuRSMKO mice. CONCLUSIONS: Our findings indicate that HuR regulates vascular smooth muscle contraction and maintains blood pressure by modulating RGS expression.

Adipose HuR protects against diet-induced obesity and insulin resistance.

Human antigen R (HuR) is a member of the Hu family of RNA-binding proteins and is involved in many physiological processes. Obesity, as a worldwide healthcare problem, has attracted more and more attention. To investigate the role of adipose HuR, we generate adipose-specific HuR knockout (HuRAKO) mice. As compared with control mice, HuRAKO mice show obesity when induced with a high-fat diet, along with insulin resistance, glucose intolerance, hypercholesterolemia and increased inflammation in adipose tissue. The obesity of HuRAKO mice is attributed to adipocyte hypertrophy in white adipose tissue due to decreased expression of adipose triglyceride lipase (ATGL). HuR positively regulates ATGL expression by promoting the mRNA stability and translation of ATGL. Consistently, the expression of HuR in adipose tissue is reduced in obese humans. This study suggests that adipose HuR may be a critical regulator of ATGL expression and lipolysis and thereby controls obesity and metabolic syndrome.

Smooth muscle-specific Gsα deletion exaggerates angiotensin II-induced abdominal aortic aneurysm formation in mice in vivo.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease without an effective pharmaceutical treatment. Genetic studies have proved the involvement of smooth muscle phenotype switch in the development of AAA. The alpha subunit of the heterotrimeric G stimulatory protein (Gsα) mediates receptor-stimulated production of cyclic adenosine monophosphate (cAMP). However, the role of smooth muscle Gsα in AAA formation remains unknown. APPROACH AND RESULTS: In this study, mice with knockout of smooth muscle-specific Gsα (GsαSMKO) were generated by cross-breeding Gsαflox/flox mice with SM22-CreERT2 transgenic mice, induced in adult mice by tamoxifen treatment. Gsα deficiency induced a smooth muscle phenotype switch from a contractile to a synthetic state. Mechanically, Gsα deletion reduced cAMP level and increased the level of human antigen R (HuR), which binds with the adenylate uridylate-rich elements of the 3' untranslated region of Krüppel-like factor 4 (KLF4) mRNA, thereby increasing the stability of KLF4. Moreover, genetic knockdown of HuR or KLF4 rescued the phenotype switch in Gsα-deficient smooth muscle cells. Furthermore, with acute infusion of angiotensin II, the incidence of AAA was markedly higher in ApoE-/-/GsαSMKO than ApoE-/-/Gsαflox/flox mice and induced increased elastic lamina degradation and aortic expansion. Finally, the levels of Gsα and SM α-actin were significantly lower while those of HuR and KLF4 were higher in human AAA samples than adjacent nonaneurysmal aortic sections. CONCLUSIONS: Gsα may play a protective role in AAA formation by regulating the smooth muscle phenotype switch and could be a potential therapeutic target for AAA disease.

Smooth muscle-specific LKB1 deletion exaggerates angiotensin II-induced abdominal aortic aneurysm in mice.

Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease without an effective pharmaceutical treatment. Liver kinase B1 (LKB1), a tumor suppressor, is a central regulator of cell polarity and energy homeostasis. However, the role of LKB1 in the development of AAA has not been explored. In this study, mice with knockout of smooth muscle-specific LKB1 (LKB1SMKO) were generated by cross-breeding LKB1flox/flox mice with SM22-CreERT2 transgenic mice and induced in adult mice by tamoxifen treatment. LKB1 deficiency increased the expression of matrix metalloproteinase 2 (MMP-2), which was inhibited by LKB1 overexpression. Mechanistically, LKB1 could bind to the MMP-2 transcription factor, specificity protein 1 (Sp1), thereby reducing the binding of Sp1 to the MMP-2 promoter to inhibit MMP-2 expression. LKB1 expression was significantly reduced in abdominal aortas of the mouse AAA model. Moreover, smooth muscle-specific LKB1 deletion exaggerated angiotensin II-induced AAA formation accompanied by increased AAA incidence and aortic expansion. Finally, LKB1 level was significantly lower and MMP-2 level higher in human AAA samples than adjacent nonaneurysmal aortic sections. Thus, these results suggest that LKB1 may play a protective role in AAA formation by inhibiting MMP-2 expression and could be a potential therapeutic target for AAA disease.

Melatonin enhances atherosclerotic plaque stability by inducing prolyl-4-hydroxylase α1 expression.

OBJECTIVE: Melatonin, an endogenous neurohormone secreted predominately by the pineal gland, has a variety of physiological functions. However, its protective role in atherosclerosis is not clear. In this study, we sought to investigate the potential effects of melatonin in modulating atherosclerotic plaque stability in apolipoprotein E knockout (ApoE) mice. METHOD AND RESULTS: Smooth muscle cells were treated with melatonin, which significantly increased mRNA and protein levels of a key intracellular enzyme essential for collagen maturation and secretion, prolyl-4-hydroxylase α1 (P4Hα1). Mechanistically, melatonin increased Akt phosphorylation and transcriptional activation of specificity protein 1 (Sp1), which bound with the P4Hα1 promoter and then induced P4Hα1 expression. Pretreatment with either Akt inhibitor LY294002 or Sp1 inhibitor mithramycin A (MTM) could inhibit melatonin-induced P4Hα1 expression. Finally, atherosclerotic lesions were induced by placing a perivascular collar on the right common carotid artery of ApoE mice, which were received with or without different doses of melatonin or MTM. High-dose melatonin enhanced atherosclerotic plaque stability in ApoE mice in vivo by inducing the expression of P4Hα1, which was reversed by MTM. CONCLUSION: We propose that melatonin supplementation may provide a novel and promising approach to atherosclerosis treatment.

Melatonin attenuates angiotensin II-induced abdominal aortic aneurysm through the down-regulation of matrix metalloproteinases.

Abdominal aortic aneurysm (AAA) affects more than 5% of the population in developed countries and the pharmacotherapies for AAA are limited. Here, we explored whether melatonin regulates the development of AAA. In smooth muscle cells, melatonin treatment decreases angiotensin II-induced matrix metalloproteinase 2 (MMP2) and MMP9 expression. Human antigen R (HuR) could bind with the adenylateuridylate-rich elements of MMP2 and MMP9 mRNAs 3' untranslated region, resulting in the increased stability of MMP2 and MMP9 mRNAs. HuR is required for angiotensin II-induced MMP2 and MMP9 expression. Moreover, melatonin suppresses angiotensin II-induced HuR expression through inhibiting NF-κB signaling, leading to decreased MMP2 and MMP9 levels. Finally, melatonin attenuates the development of AAA in ApoE-/- mice infused with angiotensin II in vivo. These data support a role of HuR in the development of AAA and possible therapeutic roles for melatonin and/or HuR inhibition in AAA.

Heterotrimeric G Stimulatory Protein α Subunit Is Required for Intestinal Smooth Muscle Contraction in Mice.

BACKGROUND & AIMS: The α subunit of the heterotrimeric G stimulatory protein (Gsa), encoded by the guanine nucleotide binding protein, α-stimulating gene (Gnas, in mice), is expressed ubiquitously and mediates receptor-stimulated production of cyclic adenosine monophosphate and activation of the protein kinase A signaling pathway. We investigated the roles of Gsa in vivo in smooth muscle cells of mice. METHODS: We performed studies of mice with Cre recombinase-mediated disruption of Gnas in smooth muscle cells (GsaSMKO and SM22-CreERT2, induced in adult mice by tamoxifen). Intestinal tissues were collected for histologic, biochemical, molecular, cell biology, and physiology analyses. Intestinal function was assessed in mice using the whole-gut transit time test. We compared gene expression patterns of intestinal smooth muscle from mice with vs without disruption of Gnas. Biopsy specimens from ileum of patients with chronic intestinal pseudo-obstruction and age-matched control biopsies were analyzed by immunohistochemistry. RESULTS: Disruption of Gnas in smooth muscle of mice reduced intestinal motility and led to death within 4 weeks. Tamoxifen-induced disruption of Gnas in adult mice impaired contraction of intestinal smooth muscle and peristalsis. More than 80% of these died within 3 months of tamoxifen exposure, with features of intestinal pseudo-obstruction characterized by chronic intestinal dilation and dysmotility. Gsa deficiency reduced intestinal levels of cyclic adenosine monophosphate and transcriptional activity of the cyclic adenosine monophosphate response element binding protein 1 (CREB1); this resulted in decreased expression of the forkhead box F1 gene (Foxf1) and protein, and contractile proteins, such as myosin heavy chain 11; actin, α2, smooth muscle, aorta; calponin 1; and myosin light chain kinase. We found decreased levels of Gsa, FOXF1, CREB1, and phosphorylated CREB1 proteins in intestinal muscle layers of patients with chronic intestinal pseudo-obstruction, compared with tissues from controls. CONCLUSIONS: Gsa is required for intestinal smooth muscle contraction in mice, and its levels are reduced in ileum biopsies of patients with chronic intestinal pseudo-obstruction. Mice with disruption of Gnas might be used to study human chronic intestinal pseudo-obstruction.

Noninvasive Regional Aortic Stiffness for Monitoring the Early Stage of Abdominal Aortic Aneurysm in Mice.

BACKGROUND: Abdominal aortic aneurysm (AAA) affects more than 5% of the population in developed countries. To study the formation and progression of AAA, we developed a non-invasive method to analyse regional aortic stiffness to monitor the formation and progression of AAA. METHODS: Saline or Angiotensin II (AngII) was subcutaneously infused in apolipoprotein E knockout (ApoE-/-) mice for 28 days; a high-resolution imaging system was used to identify changes in arterial stiffness measured by pulse-wave velocity (PWV) and aortic lumen diameter in the suprarenal aorta. RESULTS: Both regional PWV and luminal diameter in the suprarenal aorta did not change significantly in saline-treated ApoE-/- mice for 28 days. In contrast, AngII treatment for 28 days rapidly increased both regional PWV and luminal diameter. The difference in luminal diameter could be identified at 14 days. However, regional PWV significantly increased within the first 7 days after AngII perfusion as compared with saline treatment. However, in ApoE-/- diabetic mice, both regional PWV and aortic diameter did not differ between AngII and saline treatment at 7 or 28 days. CONCLUSIONS: Regional PWV may be used to monitor AAA development and was improved after AngII infusion in ApoE-/- mice.

Systematic review and meta analysis of randomized controlled trials on Tianmagouteng decoction in treatment of primary hypertension with liver Yang hyperactivity syndrome.

OBJECTIVE: To assess the efficacy of Tianmagouteng decoction in the treatment of primary hypertension with liver yang hyperactivity syndrome. METHODS: The databases of MEDLINE (1950-2012), EMbase (1980-2012), China biology medicine (CBM) (1994-2012), China national knowledge infrastructure (CNKI) (1989-2012), Weipu (VIP) (1995-2012) and Wanfang (1989-2012) were searched and we performed manual searches for conference proceedings to select randomized controlled trials (RCTs) on Tianmagouteng decoction for the treatment of primary hypertension with liver Yang hyperactivity syndrome. Studies were enrolled according to the study-selecting criteria. The methodological quality was evaluated, data were extracted and meta-analyses were conducted with RevMan 4.2 software. RESULTS: Nine RCTs were selected involving 784 patients with no significant difference in decrease in systolic blood pressure (SBP) [OR = 1.02, 95% CI (-0.24, 2.29), P = 0.10] and diastolic blood pressure (DBP) [OR = 0.56, 95% CI (-0.10,1.23), P = 0.11] between the Tianmagouteng group and the control group. While there was a significantly larger increase in serum superoxide dismutase (SOD) [OR = 6.26, 95% CI (1.27,11.66), P = 0. 01] in the Tianmagouteng group than that in the control group. CONCLUSION: Tianmagouteng decoction can decrease both systolic and diastolic blood pressure and greatly increase serum SOD. Due to poor quality of included studies, the efficacy needs to be further assessed.