Transient Receptor Potential Melastatin 7 Promotes Vascular Adventitial Fibroblasts Phenotypic Transformation and Inflammatory Reaction Induced by Mechanical Stretching Stress via p38 MAPK/JNK Pathway.

Remodeling of the arteries is one of the pathological bases of hypertension. We have previously shown that transient receptor potential melastatin 7 (TRPM7) aggravates the vascular adventitial remodeling caused by pressure overload in the transverse aortic constriction (TAC) model. In this study, we sought to explore the functional expression and downstream signaling of TRPM7 in vascular adventitial fibroblasts (AFs) stimulated by mechanical stretching stress (MSS). The expression of TRPM7 was upregulated with a concomitant translocation to the cytoplasm in the AFs stimulated with 20% MSS. Meanwhile, the expression of α-smooth muscle actin (α-SMA), a marker of transformation from AFs to myofibroblasts (MFs) was also increased. Moreover, AF-conditioned medium caused a significant migration of macrophages after treatment with MSS and contained high levels of monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α). Pharmacological and RNA interference approaches using the TRPM7 inhibitor 2-aminoethoxydiphenyl borate (2-APB) and specific anti-TRPM7 small interfering RNA (si-RNA-TRPM7) abrogated these changes significantly. Further exploration uncloaked that inhibition of TRPM7 reduced the phosphorylation of p38 MAP kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) in the AFs stimulated with MSS. Furthermore, inhibition of the phosphorylation of p38MAPK or JNK could also alleviate the MSS-induced expression of α-SMA and secretion of inflammatory factors. These observations indicate that activated TRPM7 participates in the phenotypic transformation and inflammatory action of AFs in response to MSS through the p38MAPK/JNK pathway and suggest that TRPM7 may be a potential therapeutic target for vascular remodeling caused by hemodynamic changes in hypertension.

Transactivation domain of Krüppel-like factor 15 negatively regulates angiotensin II-induced adventitial inflammation and fibrosis.

Krüppel-like factor (KLF) 15 has emerged as a critical regulator of fibrosis in cardiovascular diseases. However, the precise role that KLF15 and its functional domain played in adventitial inflammation and fibrosis remains unclear. This study aims to investigate the role of the transactivation domain (TAD) of KLF15 in angiotensin II (Ang II)-induced adventitial pathologic changes. KLF15 expression was decreased in the vascular adventitia of Ang II-infused mice (1000 ng/kg/min, 14 d) and in adventitial fibroblasts (AFs) stimulated by Ang II (10-7 M). Adenovirus-mediated KLF15 overexpression normalized Ang II-induced vascular hypertrophy, increased collagen deposition, macrophage infiltration, and CCL2 and VCAM-1 expression. Interestingly, KLF15-ΔTAD (KLF15 with deletion of TAD at amino acids 132-152) overexpression showed no effect on the above pathologic changes. Similarly, perivascularly overexpression of KLF15 but not KLF15-ΔTAD in carotid arteries also attenuated Ang II-induced vascular inflammation and fibrosis. Furthermore, KLF15 overexpression after Ang II infusion rescued Ang II-induced vascular remodeling. CCL2 or VCAM-1-mediated monocyte and macrophage migration or adhesion to AFs in response to Ang II was negatively regulated by KLF15 through TAD. Ang II-enhanced Smad2/3 activation and adventitial migration, proliferation, and differentiation of AFs were suppressed by KLF15 but not KLF15-ΔTAD overexpression. Conversely, small interfering RNA knockdown of KLF15 aggravated Ang II-induced Smad2/3 activation and dysfunction of AFs. Luciferase, coimmunoprecipitation, and chromatin immunoprecipitation assay were used to demonstrate that interaction of KLF15 with Smad2/3 suppressed CCL2 expression through TAD. Mechanistically, activation of Ang II type 1 receptor/phospholipase Cγ 1/ERK1/2 signaling resulted in a decrease of KLF15 expression. In conclusion, these results demonstrate that KLF15 negatively regulates activation of AFs through TAD, which plays an important role in Ang II-induced adventitial inflammation and fibrosis.-Lu, Y.-Y., Li, X.-D., Zhou, H.-D., Shao, S., He, S., Hong, M.-N., Liu, J.-C., Xu, Y.-L., Wu, Y.-J., Zhu, D.-L., Wang, J.-G., Gao, P.-J. Transactivation domain of Krüppel-like factor 15 negatively regulates angiotensin II-induced adventitial inflammation and fibrosis.

Deficiency of Complement C3a and C5a Receptors Prevents Angiotensin II-Induced Hypertension via Regulatory T Cells.

RATIONALE: Inflammation and immunity play crucial roles in the development of hypertension. Complement activation-mediated innate immune response is involved in the regulation of hypertension and target-organ damage. However, whether complement-mediated T-cell functions could regulate blood pressure elevation in hypertension is still unclear. OBJECTIVE: We aim to determine whether C3aR (complement component 3a receptor) and C5aR (complement component 5a receptor) could regulate blood pressure via modulating regulatory T cells (Tregs). METHODS AND RESULTS: We showed that angiotensin II (Ang II)-induced hypertension resulted in an elevated expression of C3aR and C5aR in Foxp3 (forkhead box P3)+ Tregs. By using C3aR and C5aR DKO (double knockout) mice, we showed that C3aR and C5aR deficiency together strikingly decreased both systolic and diastolic blood pressure in response to Ang II compared with WT (wild type), single C3aR-deficient (C3aR-/-), or C5aR-deficient (C5aR-/-) mice. Flow cytometric analysis showed that Ang II-induced Treg reduction in the kidney and blood was also blocked in DKO mice. Histological analysis indicated that renal and vascular structure remodeling and damage after Ang II treatment were attenuated in DKO mice compared with WT mice. In vitro, Ang II was able to stimulate C3aR and C5aR expression in cultured CD4+CD25+ natural Tregs. CD3 and CD28 antibody stimuli downregulated Foxp3 expression in WT but not DKO Tregs. More important, depletion of Tregs with CD25 antibody abolished the protective effects against Ang II-induced hypertension and target-organ damage in DKO mice. Adoptive transfer of DKO Tregs showed much more profound protective effects against Ang II-induced hypertension than WT Treg transfer. Furthermore, we demonstrated that C5aR expression in Foxp3+ Tregs was higher in hypertensive patients compared with normotensive individuals. CONCLUSIONS: C3aR and C5aR DKO-mediated Treg function prevents Ang II-induced hypertension and target-organ damage. Targeting C3aR and C5aR in Tregs specifically may be an alternative novel approach for hypertension treatment.

Developmental and functional characteristics of the thoracic aorta perivascular adipocyte.

Thoracic aorta perivascular adipose tissue (T-PVAT) has critical roles in regulating vascular homeostasis. However, the developmental characteristics and cellular lineage of adipocyte in the T-PVAT remain unclear. We show that T-PVAT contains three long strip-shaped fat depots, anterior T-PVAT (A-T-PVAT), left lateral T-PVAT (LL-T-PVAT), and right lateral T-PVAT (RL-T-PVAT). A-T-PVAT displays a distinct transcriptional profile and developmental origin compared to the two lateral T-PVATs (L-T-PVAT). Lineage tracing studies indicate that A-T-PVAT adipocytes are primarily derived from SM22α+ progenitors, whereas L-T-PVAT contains both SM22α+ and Myf5+ cells. We also show that L-T-PVAT contains more UCP1+ brown adipocytes than A-T-PVAT, and L-T-PVAT exerts a greater relaxing effect on aorta than A-T-PVAT. Angiotensin II-infused hypertensive mice display greater macrophage infiltration into A-T-PVAT than L-T-PVAT. These combined results indicate that L-T-PVAT has a distinct development from A-T-PVAT with different cellular lineage, and suggest that L-T-PVAT and A-T-PVAT have different physiological and pathological functions.

Association of ATP2B1 common variants with asymptomatic intracranial and extracranial large artery stenosis in hypertension patients.

BACKGROUND AND AIMS: Genetic factors play an important role in the cervico-cerebral large-artery atherosclerotic stenosis (LAS), and ATP2B1 gene has been associated with the process of atherosclerosis disorders, such as coronary artery disease and arterial stiffness. But there is little information about the relationship between ATP2B1 gene and atherosclerosis in the intracranial arteries. We hereby investigated the association of common variants in ATP2B1 gene with LAS in asymptomatic Chinese hypertension patients. METHODS: The stenosis of intracranial and extracranial arteries were evaluated in 899 subjects through computerized tomography angiography from the aortic arch to the skull base. A total of 11 ATP2B1 common variants were genotyped. Multivariate logistic regression was carried out in a dominant model with confounding factors adjusted. RESULTS: rs17249754-A (OR = 0.43, p = 0.0002) and rs1401982-G (OR = 0.47, p = 0.0007) were associated with decreased susceptibility of concurrent extra and intracranial stenosis even after Bonferroni correction. These two minor alleles were also significantly associated with less stenotic arteries and moderate-to-severe stenosis. CONCLUSION: rs17249754 and rs1401982 were associated with asymptomatic LAS in stroke-free Chinese hypertension patients and might benefit early recognition of LAS patients in clinical practice.

Complement-mediated inhibition of adiponectin regulates perivascular inflammation and vascular injury in hypertension.

Perivascular adipose tissue (PVAT)-derived adiponectin (APN) is a secreted adipokine that protects against hypertension-related cardiovascular injury. However, the regulation of APN expression in hypertension remains to be explored. In this study, we demonstrated that down-regulation of APN was associated with complement activation in the PVAT of desoxycorticosterone acetate (DOCA)-salt hypertensive mice. Complement 3-deficient hypertensive mice were protected from ANP decrease in the PVAT. APN deficiency blockaded the protective effects of complement inhibition against hypertensive vascular injury. Mechanistically, complement 5a (C5a)-induced TNF-α secretion from macrophages is required for inhibiting APN expression in adipocytes. Macrophage depletion reversed C5a agonist peptide-induced TNF-α up-regulation and APN down-regulation in the PVAT of DOCA mice. Moreover, we detected increased macrophage infiltration and C5a expression associated with decreased APN expression in adipose tissue from patients with aldosterone-producing adenoma. These results identify a novel interaction between macrophages and adipocytes in the PVAT, where complement-mediated inhibition of APN acts as a potential risk factor for hypertensive vascular inflammation.-Ruan, C.-C., Ma, Y., Ge, Q., Li, Y., Zhu, L.-M., Zhang, Y., Kong, L.-R., Wu, Q-H., Li, F., Cheng, L., Zhao, A. Z., Zhu, D.-L., Gao, P.-J. Complement-mediated inhibition of adiponectin regulates perivascular inflammation and vascular injury in hypertension.

[Effect of Arg188Gln (G/A) mutation on enzymatic activity of kynureninase].

Objective: To verify whether the enzymatic activity of kynureninase (KYNU) could be changed by the Arg188Gln (G/A) mutation. Methods: The total RNA of human hepatic tissue was extracted and the KYNU gene cDNA was amplified by RT-PCR. Primers were designed according to the sequences around the site Arg188Gln of KYNU gene and the Arg188Gln (G/A) mutant KYNU cDNA was generated by site-directed mutagenesis. Both the wild-type and mutant-type KYNU genes were subcloned into pcDNA vectors and the recombinant plasmids were constructed. After being transfected into human embryonic kidney 293 (HEK293) cells, the expression of KYNU recombinant plasmids were assessed by Western blot. The enzymatic activities of KYNU were detected by high performance liquid chromatography (HPLC). Results: The KYNU enzyme activities were expressed in both wild and mutant HEK293 cells. Michaelis constants (Km) of the wild and mutant KYNU were (9.833±0.513) µmol/L and (29.900±0.265) µmol/L, respectively (P<0.05). The maximum velocities (Vmax) of the wild and mutant KYNU were (0.700±0.096) nmol·mg-1·min-1 and (0.084±0.003) nmol·mg-1·min-1, respectively (P<0.05). Conclusion: Arg188Gln (G/A) mutation can decrease the enzymatic activity of KYNU.

Macrophages activate iNOS signaling in adventitial fibroblasts and contribute to adventitia fibrosis.

A large amount of NO is generated through the inducible nitric oxide synthase (iNOS) pathway from the vascular adventitia in various vascular diseases. However, it is currently not fully understood how the iNOS signaling pathway is activated. In the present study, this question was addressed in the context of adventitial cellular interactions. A rat model of acute hypertension in the contralateral carotid arteries was established through transverse aortic constriction (TAC) surgery. In this model, activated macrophages were found surrounded by a large quantity of iNOS-expressing adventitial fibroblasts (AFs), suggesting a possible causal relationship between macrophages and iNOS activation of the neighboring AFs. In an in vitro model, a macrophage-like cell line RAW 264.7 was first activated by LPS treatment. The supernatant was then harvested and applied to treat primary rat AFs. iNOS in AFs was activated robustly by the supernatant treatment but not by LPS itself. Treating AFs with interleukin-1ß (IL-1ß) also activated iNOS signaling, suggesting that the IL-1ß pathway might be a possible mediator. As a consequence of the iNOS activation, total protein nitration and S-nitrosylation significantly increased in those AFs. Additionally, increased deposition of type I and type III collagens was observed in both in vitro and in vivo models. The collagen deposition was partially restored by an iNOS inhibitor, 1400 W. These findings highlight the importance of iNOS signaling during vascular inflammation, and advance our understanding of its activation through a cellular interaction perspective.

Complement-mediated macrophage polarization in perivascular adipose tissue contributes to vascular injury in deoxycorticosterone acetate-salt mice.

OBJECTIVE: We have previously shown an increased expression of complement 3 (C3) in the perivascular adipose tissue (PVAT) in the deoxycorticosterone acetate (DOCA)-salt hypertensive model. This study aims to examine the role and underlying mechanism of C3 in PVAT for understanding the pathogenesis of hypertensive vascular remodeling further. APPROACH AND RESULTS: The role of C3 in macrophage polarization was investigated using peritoneal macrophages from wild-type and C3-deficient (C3KO) mice because we found that C3 was primarily expressed in macrophages in PVAT of blood vessels from DOCA-salt mice, and results showed a decreased expression of M1 phenotypic marker in contrast to an increased level of M2 marker in the C3KO macrophages. Bone marrow transplantation studies further showed in vivo that DOCA-salt recipient mice had fewer M1 but more M2 macrophages in PVAT when the donor bone marrows were from C3KO compared with those from wild-type mice. Of note, this macrophage polarization shift was accompanied with an ameliorated vascular injury. Furthermore, we identified the complement 5a (C5a) as the major C3 activation product that was involved in macrophage polarization and DOCA-salt-induced vascular injury. Consistently, in vivo depletion of macrophages prevented the induction of C3 and C5a in PVAT, and ameliorated hypertensive vascular injury as well. CONCLUSIONS: The presence and activation of bone marrow-derived macrophages in PVAT are crucial for complement activation in hypertensive vascular inflammation, and C5a plays a critical role in DOCA-salt-induced vascular injury by stimulating macrophage polarization toward a proinflammatory M1 phenotype in PVAT.

Rare SNP rs12731181 in the miR-590-3p Target Site of the Prostaglandin F2α Receptor Gene Confers Risk for Essential Hypertension in the Han Chinese Population.

OBJECTIVE: To investigate whether rs12731181 (A→G) interrupted miR-590-3p-mediated suppression of the prostaglandin F2α receptor (FP) and whether it is associated with essential hypertension in the Chinese population. APPROACH AND RESULTS: We found that miR-590-3p regulates human FP gene expression by binding to its 3'-untranslated region. rs12731181 (A→G) altered the binding affinity between miR-590-3p and its FP 3'-untranslated region target, thus reducing the suppression of FP expression, which, in turn, enhanced FP receptor-mediated contractility of vascular smooth muscle cells. Overexpression of FP augmented vascular tone and elevated blood pressure in mice. An association study was performed to analyze the relationship between the FP gene and essential hypertension in the Han Chinese population. The results indicated that the rs12731181 G allele was associated with susceptibility to essential hypertension. Carriers of the AG genotype exhibited significantly higher blood pressure than those of the AA genotype. FP gene expression was significantly higher in human peripheral leukocytes from individuals with the AG genotype than that in leukocytes from individuals with the AA genotype. CONCLUSIONS: rs12731181 in the seed region of the miR-590-3p target site is associated with increased risk of essential hypertension and represents a new paradigm for FP involvement in blood pressure regulation.

Association of Brachial-Ankle Pulse Wave Velocity with Asymptomatic Intracranial Arterial Stenosis in Hypertension Patients.

BACKGROUND: Intracranial arterial stenosis is a common cause of ischemic stroke in Asians. We therefore sought to explore the relationship of brachial-ankle pulse wave velocity and intracranial arterial stenosis in 834 stroke-free hypertensive patients. METHODS: Intracranial arterial stenosis was evaluated through computerized tomographic angiography. Brachial-ankle pulse wave velocity was measured by an automated cuff device. RESULTS: The top decile of brachial-ankle pulse wave velocity was significantly associated with intracranial arterial stenosis (P = .027, odds ratio = 1.82; 95% confidence interval: 1.07-3.10). The patients with the top decile of brachial-ankle pulse wave velocity showed 56% higher risk for the presence of intracranial arterial stenosis to the whole population, which was more significant in patients younger than 65 years old. We also found that brachial-ankle pulse wave velocity related to both intracranial arterial stenosis and homocysteine. CONCLUSION: Our study showed the association of brachial-ankle pulse wave velocity with asymptomatic intracranial arterial stenosis in hypertension patients, especially in relative younger subjects. Brachial-ankle pulse wave velocity might be a relatively simple and repeatable measurement to detect hypertension patients in high risk of intracranial arterial stenosis.

Deletion of angiotensin-converting enzyme 2 exacerbates renal inflammation and injury in apolipoprotein E-deficient mice through modulation of the nephrin and TNF-alpha-TNFRSF1A signaling.

BACKGROUND: The renin-angiotensin system (RAS) has been implicated in atherosclerotic lesions and progression to chronic kidney diseases. We examined regulatory roles of angiotensin-converting enzyme 2 (ACE2) in the apolipoprotein E (ApoE) knockout (KO) kidneys. METHODS: The 3-month-old wild-type, ApoEKO, ACE2KO and ApoE/ACE2 double-KO (DKO) mice in a C57BL/6 background were used. The ApoEKO mice were randomized to daily deliver either Ang II (1.5 mg/kg) and/or human recombinant ACE2 (rhACE2; 2 mg/kg) for 2 weeks. We examined changes in pro-inflammatory cytokines, renal ultrastructure, and pathological signaling in mouse kidneys. RESULTS: Downregulation of ACE2 and nephrin levels was observed in ApoEKO kidneys. Genetic ACE2 deletion resulted in modest elevations in systolic blood pressure levels and Ang II type 1 receptor expression and reduced nephrin expression in kidneys of the ApoE/ACE2 DKO mice with a decrease in renal Ang-(1-7) levels. These changes were linked with marked increases in renal superoxide generation, NADPH oxidase (NOX) 4 and proinflammatory factors levels, including interleukin (IL)-1beta, IL-6, IL-17A, RANTES, ICAM-1, Tumor necrosis factor-alpha (TNF-alpha) and TNFRSF1A. Renal dysfunction and ultrastructure injury were aggravated in the ApoE/ACE2 DKO mice and Ang II-infused ApoEKO mice with increased plasma levels of creatinine, blood urea nitrogen and enhanced levels of Ang II in plasma and kidneys. The Ang II-mediated reductions of renal ACE2 and nephrin levels in ApoEKO mice were remarkably rescued by rhACE2 supplementation, along with augmentation of renal Ang-(1-7) levels. More importantly, rhACE2 treatment significantly reversed Ang II-induced renal inflammation, superoxide generation, kidney dysfunction and adverse renal injury in ApoEKO mice with suppression of the NOX4 and TNF-alpha-TNFRSF1A signaling. However, rhACE2 had no effect on renal NOX2 and TNFRSF1B expression and circulating lipid levels. CONCLUSIONS: ACE2 deficiency exacerbates kidney inflammation, oxidative stress and adverse renal injury in the ApoE-mutant mice through modulation of the nephrin, NOX4 and TNF-alpha-TNFRSF1A signaling. While rhACE2 supplementation alleviates inflammation, renal dysfunction and glomerulus injury in the ApoE-mutant mice associated with upregulations of Ang-(1-7) levels and nephrin expression and suppression of the TNF-alpha-TNFRSF1A signaling. Strategies aimed at enhancing the ACE2/Ang-(1-7) actions may have important therapeutic potential for atherosclerotic renal injury and kidney diseases.

An Evaluation of Laboratory Efficiency in Shanghai Emergency by Turn Around Times Level.

OBJECTIVE: China launched a health care reform policy due to the aging population and rapid urbanization. However, emergency overcrowding is not improved. We assessed the laboratory efficiency of emergency department (ED) in Shanghai hospitals. METHODS: We recorded the turn around times for processing laboratory biomarkers to assess laboratory efficiency at 17 EDs in national/regional hospitals. We compared TAT between national and regional hospitals and between central and ED laboratories to analyze the relationship between the laboratory efficiency and the ED overcrowding. RESULTS: All the participating hospitals have an emergency laboratory. The median TAT for c-TNT was 61 min (46-76 min) at regional EDs compared with 64 min (46-87 min) at national EDs; therefore, the TAT at regional EDs were more efficient (P < 0.05). The TAT were longer (65 min (53-85 min)) at ED labs than (60 min (42-83 min)) at central labs (P < 0.05), independent of the hospital tier and working period. We discovered that only 9% of investigated samples at Tier II EDs and 5% at Tier III EDs were assayed by point-of-care (POC) instruments. CONCLUSION: Our TAT level is approaching the recommended international standard. However, the TAT evaluation from ED laboratories demonstrates that their existence does not decrease the waiting time for laboratory reports compared to central laboratory. Thus, they have not yet approached a level to share the burden of the ED overcrowding. Further arrangement should be assigned to separate the function of emergency laboratory and central laboratory. It is worth deploying the POC assay in the ED, which will save twice the TAT level. The idea of evaluating routine laboratory efficiency by TAT at ED is fast, convenient, although it does not represent the general level of laboratory efficiency.

Efficacy of folic acid therapy in primary prevention of stroke among adults with hypertension in China: the CSPPT randomized clinical trial.

IMPORTANCE: Uncertainty remains about the efficacy of folic acid therapy for the primary prevention of stroke because of limited and inconsistent data. OBJECTIVE: To test the primary hypothesis that therapy with enalapril and folic acid is more effective in reducing first stroke than enalapril alone among Chinese adults with hypertension. DESIGN, SETTING, AND PARTICIPANTS: The China Stroke Primary Prevention Trial, a randomized, double-blind clinical trial conducted from May 19, 2008, to August 24, 2013, in 32 communities in Jiangsu and Anhui provinces in China. A total of 20,702 adults with hypertension without history of stroke or myocardial infarction (MI) participated in the study. INTERVENTIONS: Eligible participants, stratified by MTHFR C677T genotypes (CC, CT, and TT), were randomly assigned to receive double-blind daily treatment with a single-pill combination containing enalapril, 10 mg, and folic acid, 0.8 mg (n = 10,348) or a tablet containing enalapril, 10 mg, alone (n = 10,354). MAIN OUTCOMES AND MEASURES: The primary outcome was first stroke. Secondary outcomes included first ischemic stroke; first hemorrhagic stroke; MI; a composite of cardiovascular events consisting of cardiovascular death, MI, and stroke; and all-cause death. RESULTS: During a median treatment duration of 4.5 years, compared with the enalapril alone group, the enalapril-folic acid group had a significant risk reduction in first stroke (2.7% of participants in the enalapril-folic acid group vs 3.4% in the enalapril alone group; hazard ratio [HR], 0.79; 95% CI, 0.68-0.93), first ischemic stroke (2.2% with enalapril-folic acid vs 2.8% with enalapril alone; HR, 0.76; 95% CI, 0.64-0.91), and composite cardiovascular events consisting of cardiovascular death, MI, and stroke (3.1% with enalapril-folic acid vs 3.9% with enalapril alone; HR, 0.80; 95% CI, 0.69-0.92). The risks of hemorrhagic stroke (HR, 0.93; 95% CI, 0.65-1.34), MI (HR, 1.04; 95% CI, 0.60-1.82), and all-cause deaths (HR, 0.94; 95% CI, 0.81-1.10) did not differ significantly between the 2 treatment groups. There were no significant differences between the 2 treatment groups in the frequencies of adverse events. CONCLUSIONS AND RELEVANCE: Among adults with hypertension in China without a history of stroke or MI, the combined use of enalapril and folic acid, compared with enalapril alone, significantly reduced the risk of first stroke. These findings are consistent with benefits from folate use among adults with hypertension and low baseline folate levels. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00794885.

The association of serum inflammatory biomarkers with chronic kidney disease in hypertensive patients.

A positive association between inflammation and chronic kidney disease (CKD) has been reported but the impact of hypertension on this relation remains unclear. The aim of this study is to investigate the association of various inflammation markers with risk of CKD in hypertensive patients. 387 hypertensive patients (mean age 55.5 years) were recruited. Serum matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1(TIMP-1), high-sensitivity C-reactive protein (hsCRP) and osteopontin (OPN) were measured by ELISA. CKD was diagnosed either as evidence of kidney damage, including microalbuminuria, or by low glomerular filtration rate (GFR) (<60 mL/min/1.73 m(2)), which was estimated using the Modification of Diet in Renal Disease (MDRD) abbreviated equation. Compared with the reference groups (eGFR ≥ 60 mL/min/1.73 m(2)), the serum levels of TIMP-1, OPN, hsCRP were significantly higher, and the MMP-9/TIMP-1 ratio was lower in the risk group (eGFR < 60 mL/min/1.73 m(2)). Multiple logistic regression analysis showed that TIMP-1, MMP-9/TIMP-1 ratio, OPN and hsCRP were associated with low GFR separately after adjustment, whereas MMP-9/TIMP-1 ratio, OPN and hsCRP were associated with microalbuminuria. The significant association of MMP-9/TIMP-1 ratio and OPN with low GFR and microalbuminuria persisted after additional adjustment for other studied inflammatory biomarkers. Our data suggest that inflammation is strongly and independently associated with renal damage in hypertensive patients. MMP-9/TIMP-1 ratio and OPN may serve as novel risk factors and therapeutic targets for the treatment of CKD in hypertensive patients.

Intelligent Assist Office Blood Pressures (IOBP) versus awake ambulatory monitoring and conventional auscultatory office readings in Chinese primary medical institutions.

To practice standardized office blood pressure (OBP) measurement guidelines pragmatically, we developed an intelligent assisted OBP (IOBP) measurement system in the Chinese community, which can automatically obtain two or three BP values after a 5-min rest before the patients visit the doctor and transfer values to the community medical network in real time. We conducted a comparative study to investigate the agreement among IOBP, awake ambulatory BP (ABP), and conventional auscultatory OBP at different BP levels. Participants were divided into three groups according to BP, with 120/80 mmHg and 160/100 mmHg as the cut-off points. Attended IOBP, unattended IOBP, and auscultatory OBP were randomly measured before ABP monitoring. In total, 245 participants were included in the analysis. The mean systolic attended/unattended IOBP, auscultatory OBP, and awake ABP were 135.0, 136.7, 135.6, and 136.2 mmHg, respectively. Bland-Altman analysis revealed a bias of -1.1 and 0.5 mmHg for systolic attended/unattended IOBP compared with awake ABP in the overall sample. For auscultatory OBP, the bias was -0.4 (attended) and 1.2 mmHg (unattended). The discrepancy between the systolic attended/unattended IOBP and awake ABP was inconsistent at different BP levels. In Group 1 the values were -8.4 and -6.9 mmHg, whereas in Group 3, the values were 9.4 and 10.0 mmHg. BP measured using the IOBP measurement system was in accordance with awake ABP and conventional OBP, and can be a good choice in the Chinese community.

Cardiac protective effects of irbesartan via the PPAR-gamma signaling pathway in angiotensin-converting enzyme 2-deficient mice.

BACKGROUND: Angiotensin-converting enzyme 2 (ACE2), a monocarboxypeptidase which metabolizes angiotensin II (Ang II) to generate Ang-(1-7), has been shown to prevent cardiac hypertrophy and injury but the mechanism remains elusive. Irbesartan has the dual actions of angiotensin receptor blockade and peroxisome proliferator-activated receptor-γ (PPARγ) activation. We hypothesized that irbesartan would exert its protective effects on ACE2 deficiency-mediated myocardial fibrosis and cardiac injury via the PPARγ signaling. METHODS: 10-week-old ACE2 knockout (ACE2KO; Ace2(-/y)) mice received daily with irbesartan (50 mg/kg) or saline for 2 weeks. The wild-type mice (Ace2(+/y)) were used to the normal controls. We examined changes in myocardial ultrastructure, fibrosis-related genes and pathological signaling by real-time PCR gene array, Western blotting, Masson trichrome staining and transmission electron microscope analyses, respectively. RESULTS: Compared with the Ace2(+/y) mice, cardiac expression of PPARα and PPARγ were reduced in Ace2(-/y) mice and the myocardial collagen volume fraction (CVF) and expression of fibrosis-related genes were increased, including transforming growth factor-ß1 (TGFß1), connective tissue growth factor (CTGF), collagen I and collagen III. Moreover, ACE2 deficiency triggered cardiac hypertrophy, increased myocardial fibrosis and adverse ultrastructure injury in ACE2KO hearts with higher levels of atrial natriuretic factor (ANF) and phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), without affecting cardiac systolic function. Intriguingly, treatment with irbesartan significantly reversed ACE2 deficiency-mediated pathological hypertrophy and myocardial fibrosis in Ace2(-/y) mice linked with enhancement of plasma Ang-(1-7) level and downregulation of AT1 receptor in heart. Consistent with attenuation of myocardial fibrosis and ultrastructure injury, the myocardial CVF and levels of ANF, TGFß1, CTGF, collagen I, collagen III and phosphorylated ERK1/2 were lower, and expression of PPARγ was higher in ACE2KO mice in response to irbesartan treatment, without affecting cardiac expression of PPARα, PPARδ, ß-myosin heavy chain, TGFß2 and fibronectin. CONCLUSIONS: We conclude that irbesartan prevents ACE2 deficiency-mediated pathological hypertrophy and myocardial fibrosis in ACE2 mutant mice via activation of the PPARγ signaling and suppression of the TGFß-CTGF-ERK signaling, resulting in attenuation of myocardial injury. Drugs targeting ACE2 and PPARγ represent potential candidates to prevent and treat myocardial injury and related cardiac disorders.

Vascular endothelial growth factor-induced osteopontin expression mediates vascular inflammation and neointima formation via Flt-1 in adventitial fibroblasts.

OBJECTIVE: Adventitia acts as an active participant in vascular inflammation but the precise mechanism underlying adventitia-mediated vascular inflammation is not fully understood. In this study, we sought to determine whether vascular endothelial growth factor (VEGF) regulates osteopontin (OPN) expression through Flt-1 in adventitial fibroblasts (AFs) to mediate vascular inflammation and neointima formation. METHODS AND RESULTS: In primary cultured AFs, VEGF increased intracellular and secreted OPN expression in a time- and dose-dependent manner, which was effectively suppressed by a specific anti-Flt-1 hexapeptide. Interestingly, VEGF treatment of AFs enhanced the capability of AF-conditioned medium to stimulate macrophages chemotaxis, and this effect was attenuated after blockade of OPN from AF-conditioned medium. Furthermore, perivascular delivery of anti-Flt-1 peptide preferentially concentrated in the adventitia resulted in a decrease of neointima formation after balloon injury in carotid arteries. The inhibition of neointima formation was preceded by significant reduction of VEGF and OPN expression with concurrent macrophage infiltration into adventitia after injury. Activation of extracellular signal-regulated kinase 1/2 pathway was involved in OPN upregulation and macrophage chemotaxis. CONCLUSIONS: These results demonstrate that VEGF/Flt-1 signaling plays a significant role in vascular inflammation and neointima formation by regulating OPN expression in AFs and provide insight into Flt-1 as a potential therapeutic target for vascular diseases.

Loss of angiotensin-converting enzyme 2 exacerbates myocardial injury via activation of the CTGF-fractalkine signaling pathway.

BACKGROUND: Angiotensin-converting enzyme 2 (ACE2) has been implicated in human heart failure, but the mechanism remains elusive. We hypothesized that ACE2 deficiency would exacerbate angiotensin (Ang) II-mediated myocardial injury. METHODS AND RESULTS: 10-week-old ACE2 knockout (ACE2KO) and wild-type mice received by mini-osmotic pump either AngII (1.5 mg·kg(-1)·day(-1)) or saline for 2 weeks. ACE2 deficiency triggered greater increases in the expression of connective tissue growth factor (CTGF), fractalkine (FKN) and phosphorylated ERK1/2 in AngII-treated ACE2KO hearts. These changes were associated with greater activation of matrix metalloproteinase (MMP) 2, MMP9 and MT1-MMP and exacerbation of myocardial injury and dysfunction. In cultured cardiofibroblasts, exposure to AngII (100 nmol/L) for 30 min resulted in marked increases in superoxide production and expression of CTGF, FKN and phosphorylated ERK1/2, which were strikingly prevented by recombinant human ACE2 (rhACE2; 1mg/ml) and the CTGF-neutralizing antibody (5 µg/ml), but were aggravated by ACE2 inhibitor DX600 (0.5 µmol/L). These protective effects of rhACE2 were eradicated by the Ang-(1-7) antagonist A779 (1 µmol/L). More intriguingly, rhACE2 treatment significantly abolished AngII-mediated increases in MMP2, MMP9 and MT1-MMP in cardiofibroblasts. CONCLUSIONS: Loss of ACE2 exacerbates AngII-mediated inflammation, myocardial injury and dysfunction in ACE2-deficient hearts via activation of the CTGF-FKN-ERK and MMP signaling. ACE2 gene may represent a potential candidate to prevent and treat myocardial injury and heart diseases.

Common variants in the ATP2B1 gene are associated with hypertension and arterial stiffness in Chinese population.

Genome-wide association studies have identified the ATP2B1 gene associated with blood pressure (BP), the evidence from large scale Chinese population was still rare. We performed the current replication study to test the association of the ATP2B1 gene and hypertension and BP in two unrelated Chinese cohorts including 2,831 unrelated individuals with hypertension and 1,987 controls in total. We also examined the influences of the ATP2B1 gene on the arterial stiffness through evaluation of carotid-femoral pulse wave velocities (cf-PWV) in 164 untreated hypertensives. The major findings of this study were that four loci--rs10858911, rs2681472, rs17249754 and rs1401982--associated with any or all of four traits: hypertension (P = 0.001-4.6E-05; odds ratio, 0.83-0.87), systolic BP (P = 0.003-0.004), diastolic BP (P = 0.002-0.003) and cf-PWV (P = 0.002-0.004). All the comparisons were adjusted for sex, age, age(2) and body mass index. We validated the association of the ATP2B1 gene and susceptibility to hypertension, BP traits and cf-PWV in Chinese population. In addition, further genetic and functional research was warranted to elucidate the concrete locus in the ATP2B1 gene that influenced the manifestation of BP and vascular function.