C-atrial natriuretic peptide (ANP)4-23 attenuates renal fibrosis in deoxycorticosterone-acetate-salt hypertensive mice.

Epithelial-mesenchymal transition (EMT) plays a critical role in hypertension-induced renal fibrosis, a final pathway that leads to end-stage renal failure. C-Atrial natriuretic peptide (ANP)4-23, a specific agonist of natriuretic peptide receptor-C (NPR-C), has been reported to have protective effects against hypertension. However, the role of C-ANP4-23 in hypertension-associated renal fibrosis has not yet been elucidated. In this study, mice were randomly divided into SHAM group, DOCA-salt group and DOCA-salt + C-ANP4-23 group. Renal morphology changes, renal function and fibrosis were detected. Human proximal tubular epithelial cells (HK2) stimulated by aldosterone were used for cell function and mechanism study. The DOCA-salt treated mice exhibited hypertension, kidney fibrosis and renal dysfunction, which were attenuated by C-ANP4-23. Moreover, C-ANP4-23 inhibited DOCA-salt treatment-induced renal EMT as evidenced by decrease of the mesenchymal marker alpha-smooth muscle actin (ACTA2) and vimentin and increase of epithelial cell marker E-cadherin. In HK2 cells, aldosterone induced EMT response, which was also suppressed by C-ANP4-23. The key transcription factors (twist, snail, slug and ZEB1) involved in EMT were increased in the kidney of DOCA-salt-treated mice, which were also suppressed by C-ANP4-23. Mechanistically, C-ANP4-23 inhibited the aldosterone-induced translocation of MR from cytosol to nucleus without change of MR expression. Furthermore, C-ANP4-23 rescued the enhanced expression of NADPH oxidase (NOX) 4 and oxidative stress after aldosterone stimulation. Aldosterone-induced Akt and Erk1/2 activation was also suppressed by C-ANP4-23. Our data suggest that C-ANP4-23 attenuates renal fibrosis, likely through inhibition of MR activation, enhanced oxidative stress and Akt and Erk1/2 signaling pathway.

Mitogen-Activated Protein Kinases Mediate Adventitial Fibroblast Activation and Neointima Formation via GATA4/Cyclin D1 Axis.

PURPOSE: Activation of mitogen-activated protein kinases (MAPKs) by pathological stimuli participates in cardiovascular diseases. Dysfunction of adventitial fibroblast has emerged as a critical regulator in vascular remodeling, while the potential mechanism remains unclear. In this study, we sought to determine the effect of different activation of MAPKs in adventitial fibroblast contributing to neointima formation. METHODS: Balloon injury procedure was performed in male 12-week-old Sprague-Dawley rats. After injury, MAPK inhibitors were applied to the adventitia of injured arteries to suppress MAPK activation. Adventitial fibroblasts were stimulated by platelet-derived growth factor-BB (PDGF-BB) with or without MAPK inhibitors. RNA sequencing was performed to investigate the change of pathway and cell function. Wound healing, transwell assay, and flow cytometry were used to analyze adventitial fibroblast function. RESULTS: Phosphorylation of p38, c-Jun N-terminal kinase (JNK), and extracellular regulated kinases 1/2 (ERK1/2) was increased in injured arteries after balloon injury. In primary culture of adventitial fibroblasts, PDGF-BB increased phosphorylation of p38, JNK, ERK1/2, and extracellular regulated kinase 5 (ERK5) in a short time, which was normalized by their inhibitors respectively. Compared with the injury group, perivascular administration of four MAPK inhibitors significantly attenuated neointima formation by quantitative analysis of neointimal area, intima to media (I/M) ratio, and lumen area. RNA sequencing of adventitial fibroblasts treated with PDGF-BB with or without four inhibitors demonstrated differentially expressed genes involved in multiple biological processes, including cell adhesion, proliferation, migration, and inflammatory response. Wound healing and transwell assays showed that four inhibitors suppressed PDGF-BB-induced adventitial fibroblast migration. Cell cycle analysis by flow cytometry demonstrated that JNK, ERK1/2, and ERK5 but not p38 inhibitor blocked PDGF-BB-induced G1 phase release associated with decrease expression of cell cycle protein Cyclin D1 and transcription factor GATA4. Moreover, four inhibitors decreased macrophage infiltration into adventitia and monocyte chemoattractant protein-1 (MCP-1) expression. CONCLUSION: These results suggest that MAPKs differentially regulate activation of adventitial fibroblast through GATA4/Cyclin D1 axis that participates in neointima formation.

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.

Complement 5a-mediated trophoblasts dysfunction is involved in the development of pre-eclampsia.

Pre-eclampsia (PE) is a life-threatening multisystem disorder leading to maternal and neonatal mortality and morbidity. Emerging evidence showed that activation of the complement system is implicated in the pathological processes of PE. However, little is known about the detailed cellular and molecular mechanism of complement activation in the development of PE. In this study, we reported that complement 5a (C5a) plays a pivotal role in aberrant placentation, which is essential for the onset of PE. We detected an elevated C5a deposition in macrophages and C5a receptor (C5aR) expression in trophoblasts of pre-eclamptic placentas. Further study showed that C5a stimulated trophoblasts towards an anti-angiogenic phenotype by mediating the imbalance of angiogenic factors such as soluble fms-like tyrosine kinase 1 (sFlt1) and placental growth factor (PIGF). Additionally, C5a inhibited the migration and tube formation of trophoblasts, while, C5aR knockdown with siRNA rescued migration and tube formation abilities. We also found that maternal C5a serum level was increased in women with PE and was positively correlated with maternal blood pressure and arterial stiffness. These results demonstrated that the placental C5a/C5aR pathway contributed to the development of PE by regulating placental trophoblasts dysfunctions, suggesting that C5a may be a novel therapeutic possibility for the disease.

Protease-activated receptor 1 and 2 contribute to angiotensin II-induced activation of adventitial fibroblasts from rat aorta.

Adventitial fibroblasts (AFs) can be activated by angiotensin II (Ang II) and exert pro-fibrotic and pro-inflammatory effects in vascular remodeling. Protease-activated receptor (PAR) 1 and 2 play a significant role in fibrogenic and inflammatory diseases. The present study hypothesized that PAR1 and PAR2 are involved in Ang II-induced AF activation and contribute to adventitial remodeling. We found that direct activation of PAR1 and PAR2 with PAR1-AP and PAR2-AP led to AF activation, including proliferation and differentiation of AFs, extracellular matrix synthesis, as well as production of pro-fibrotic cytokine TGF-ß and pro-inflammatory cytokines IL-6 and MCP-1. Furthermore, PAR1 and PAR2 mediated Ang II-induced AF activation, since both PAR1 and PAR2 antagonists inhibited Ang II-induced proliferation, migration, differentiation, extracellular matrix synthesis and production of pro-fibrotic and pro-inflammatory cytokines in AFs. Finally, mechanistic study showed that Ang II, via Ang II type I receptor (AT1R), upregulated both PAR1 and PAR2 expression, and transactivated PAR1 and PAR2, as denoted by internalization of both proteins. In conclusion, our results suggest that PAR1 and PAR2 play a critical role in Ang II-induced AF activation, and this may contribute to adventitia-related pathological changes.

RhoA-Rho kinase signaling pathway mediates adventitial fibroblasts differentiation to myofibroblasts induced by TGF-ß1.

Vascular adventitial fibroblasts (AF) differentiation to myofibroblasts (MF) is the critical physiopathologic feature of vascular remodeling. This study was to investigate the role of RhoA-Rho kinase signaling pathway in AF differentiation to MF induced by transforming growth factor ß1 (TGF-ß1). The results showed that TGF-ß1 up-regulated total RhoA protein expression and RhoA activity in cultured AF by Western blotting and Rho pull-down assay, respectively. TGF-ß1 up-regulated phospho-Myosin phosphatase target subunit (MYPT1, a downstream substrate of Rho kinase) expression without altering Rho kinase protein expression, indicating TGF-ß1 induced the enhancement of activity of Rho kinase. Ad-N19RhoA-hrGFP virus infection and Y27632, a specific inhibitor of Rho kinase, dose-dependently inhibited TGF-ß1-induced α-SM-actin and Calponin expression, as markers of MF differentiation. In conclusion, the RhoA-Rho kinase pathway is involved in AF differentiation to MF induced by TGF-ß1.

Intravascular ultrasound provides additional insights in the hypertensive patients with focal renal artery fibromuscular dysplasia.

To date, few study has defined the exact role or utility of intravascular ultrasound (IVUS) in the diagnosis and treatment of renal artery fibromuscular dysplasia (FMD). We investigated whether using an IVUS would provide additional insights in the hypertensive patients with focal renal artery FMD. A prospective, observational study, including all patients with focal renal artery FMD admitted to the Ruijin hospital during 6 consecutive years (2015-2021). Based on IVUS imaging, focal FMD patients were classified as two subtypes: intima-media thickening (IMT) and negative remodeling (NR) of the whole vessel. A total of 36 consecutive patients (24 ± 7, 13-39 years) with focal renal artery FMD were enrolled. Angiographic unifocal type was present in 22 (61.1%) patients and tubular type was present in 14 (38.9%) patients. Among 22 patients with unifocal, IVUS showed that 18 (81.8%) had IMT and 4 (18.2%) had NR. 14 patients with tubular, IVUS showed 3 (21.4%) had IMT and 11 (78.6%) had NR. No difference in age of onset, gender, BMI, initial BP levels were found between IMT and NR subtypes. However, hypertension cure rates of short-term (48 h after angioplasty) (76.2% vs. 26.7%, p = 0.004) and long-term (1-6years) (90.5% vs. 20.0%, p < 0.001) were higher in patients with IMT than in those with NR subtype. In present study, we described a new classification of focal renal artery FMD into IMT or NR subtype based on IVUS. Renal FMD Patients with IMT subtype were more likely to achieve cure of hypertension. We investigated whether using an IVUS would provide additional insights in the hypertensive patients with focal renal artery FMD. A new classification of focal renal artery FMD into IMT or NR subtype based on IVUS was described. Renal FMD Patients with IMT subtype were more likely to achieve cure of hypertension.

Case report: Recurrence of hypertension after renal artery angioplasty due to the progression of focal renal fibromuscular dysplasia.

Whether fibromuscular dysplasia (FMD) is a progressive disease, remains unclear. We reported a case of focal renal artery FMD that slowly progressed to a branching artery over a few years after the angioplasty without in-stent restenosis, which reconfirms that focal FMD is progressive and that such progression may be segmental. Stenting may be an option for young, risk factor-free patients with focal FMD.

Diurnal blood pressure pattern and cardiac damage in hypertensive patients with primary aldosteronism.

PURPOSE: The aim of our study was to evaluate the relationship between the 24-h blood pressure (BP) profile, plasma NT-proBNP levels and left ventricular hypertrophy (LVH) in subjects with primary aldosteronism (PA) compared to patients with essential hypertension (EH). METHODS: A total of 385 consecutive patients with PA [187 with aldosterone producing adenoma (APA) and 198 with idiopathic hyperaldosteronism (IHA)] and 385 patients with EH were matched based on age, sex, body mass index (BMI), BP values and duration of hypertension. Twenty-four-hour ambulatory BP monitoring (ABPM), plasma levels of NT-proBNP, left ventricular mass index (LVMI), and other clinical medical data were assessed in all patients. RESULTS: No differences in age, sex, BMI, clinical BP, 24-h mean BP, daytime BP, or duration of hypertension were found between groups. Nighttime systolic BP (130 ± 16 vs. 127 ± 17 mmHg, p < 0.05) and diastolic BP (82 ± 10 vs. 79 ± 11 mmHg, p < 0.01) were higher in PA patients than in EH patients. In addition, nocturnal BP decline was reduced, while median NT-proBNP (53.7 vs. 33.2 pg/ml, P < 0.001) and LVMI (113 ± 25 vs. 102 ± 26 g/m2, P < 0.001) were higher in PA patients than in EH patients. Moreover, the median NT-proBNP level was higher in APA patients than in IHA patients (68.0 vs. 42.4 pg/ml, P < 0.001). In stepwise multivariate regression analysis, LVMI was correlated with NT-proBNP, nighttime systolic BP and sex in PA patients. CONCLUSIONS: Patients with PA show higher nighttime BP and NT-proBNP levels and lower nocturnal BP decline than those with EH. In addition, higher nocturnal systolic BP has been shown to be strongly associated with cardiac damage in PA patients.

Adventitial fibroblast-derived vascular endothelial growth factor promotes vasa vasorum-associated neointima formation and macrophage recruitment.

AIMS: Adventitial vasa vasorum provides oxygen and nourishment to the vascular wall, but whether it regulates vascular disease remains unclear. We have previously shown that an increased expression of VEGF (vascular endothelial growth factor) is associated with macrophage infiltration. This study aims to determine whether adventitial fibroblast (AF)-derived VEGF increases the number of vasa vasorum contributing to neointima formation through macrophage recruitment. METHODS AND RESULTS: In rat balloon injury model, vasa vasorum count was increased particularly in the adventitia accompanied by cell proliferation and VEGF expression. Both endogenous and PKH26-labelled exogenous macrophages were mainly distributed in adventitia around vasa vasorum. Interestingly, perivascular delivery of Ranibizumab preferentially concentrated in adventitia resulted in a decrease of neointima formation with concurrent reduction of vasa vasorum count and macrophage infiltration. AFs with adenovirus-mediated VEGF over-expression delivered to the adventitia significantly enhanced these pathological changes after injury. In Tie2-cre/Rosa-LoxP-RFP mice, endothelial cells were increased in the adventitia after wire injury. By using multiphoton laser scanning microscopy, macrophage rolling, adhesion and transmigration were observed in vasa vasorum. Moreover, adoptive transfer of macrophages accelerated injury-induced neointima formation. VEGF-neutralizing antibody administration also attenuated wire injury-induced neointima formation and macrophage infiltration. In primary cultured AFs, exogenous VEGF increased VEGF expression and secretion in a time- and dose-dependent manner. AF-conditioned medium promoted endothelial cell angiogenesis, vascular cell adhesion molecule-1 expression and macrophage adhesion was blocked by VEGF-neutralizing antibody and VEGFR2 inhibitor ZM323881, which also inhibited activation of VEGFR2/ERK1/2 pathway. CONCLUSION: These results demonstrate that AF-derived VEGF plays a significant role in the increase of vasa vasorum count which is involved in macrophage recruitment and neointima formation.

Renal denervation attenuates aldosterone expression and associated cardiovascular pathophysiology in angiotensin II-induced hypertension.

The sympathetic nervous system interacts with the renin-angiotensin-aldosterone system (RAAS) contributing to cardiovascular diseases. In this study, we sought to determine if renal denervation (RDN) inhibits aldosterone expression and associated cardiovascular pathophysiological changes in angiotensin II (Ang II)-induced hypertension. Bilateral RDN or SHAM operation was performed before chronic 14-day Ang II subcutaneous infusion (200ng/kg/min) in male Sprague-Dawley rats. Bilateral RDN blunted Ang II-induced hypertension and ameliorated the mesenteric vascular dysfunction. Cardiovascular hypertrophy in response to Ang II was significantly attenuated by RDN as shown by histopathology and transthoracic echocardiography. Moreover, Ang II-induced vascular and myocardial inflammation and fibrosis were suppressed by RDN with concurrent decrease in fibronectin and collagen deposition, macrophage infiltration, and MCP-1 expression. Interestingly, RDN also inhibited Ang II-induced aldosterone expression in the plasma, kidney and heart. This was associated with the reduction of calcitonin gene-related peptide (CGRP) in the adrenal gland. Ang II promoted aldosterone secretion which was partly attenuated by CGRP in the adrenocortical cell line, suggesting a protective role of CGRP in this model. Activation of transforming growth factor-ß (TGF-ß)/Smad and mitogen-activated protein kinases (MAPKs) signaling pathway was both inhibited by RDN especially in the heart. These results suggest that the regulation of the renal sympathetic nerve in Ang II-induced hypertension and associated cardiovascular pathophysiological changes is likely mediated by aldosterone, with CGRP involvement.

Mechanisms of improved aortic stiffness by arotinolol in spontaneously hypertensive rats.

OBJECTIVES: This study investigates the effects on aortic stiffness and vasodilation by arotinolol and the underlying mechanisms in spontaneously hypertensive rats (SHR). METHODS: The vasodilations of rat aortas, renal and mesenteric arteries were evaluated by isometric force recording. Nitric oxide (NO) was measured in human aortic endothelial cells (HAECs) by fluorescent probes. Sixteen-week old SHRs were treated with metoprolol (200 mg·kg-1·d⁻¹), arotinolol (30 mg·kg-1·d⁻¹) for 8 weeks. Central arterial pressure (CAP) and pulse wave velocity (PWV) were evaluated via catheter pressure transducers. Collagen was assessed by immunohistochemistry and biochemistry assay, while endothelial nitric oxide synthase (eNOS) and eNOS phosphorylation (p-eNOS) of HAECs or aortas were analyzed by western blotting. RESULTS: Arotinolol relaxed vascular rings and the relaxations were attenuated by Nω-nitro-L-arginine methyl ester (L-NAME, NO synthase inhibitor) and the absence of endothelium. Furthermore, arotinolol-induced relaxations were attenuated by 4-aminopyridine (4-AP, Kv channels blocker). Arotinolol produced more nitric oxide compared to metoprolol and increased the expression of p-eNOS in HAECs. These results indicated that arotinolol-induced vasodilation involves endothelium-derived NO and Kv channels. The treatement with arotinolol in 8 weeks, but not metoprolol, markedly decreased CAP and PWV. Biochemistry assay and immunohistochemistry showed that aortic collagen depositions in the arotinolol groups were reduced compared with SHRs with metoprolol. Moreover, eNOS phosphorylation was significantly increased in aortinolol-treated SHR compared with SHRs with metoprolol. CONCLUSIONS: Arotinolol improves arterial stiffness in SHR, which involved in increasing NO and decreasing collagen contents in large arteries.