Qian Ge

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.

Osteopontin regulates macrophage activation and osteoclast formation in hypertensive patients with vascular calcification

Vascular calcification (VC) is a highly regulated ectopic mineral deposition process involving immune cell infiltration in the vasculatures, which has been recognized to be promoted by hypertension. The matricellular glycoprotein osteopontin (OPN) is strongly induced in myeloid cells as a potential inflammatory mediator of vascular injury. This study aims to examine whether OPN is involved in the regulation of macrophage activation and osteoclast formation in hypertensive subjects with VC. We firstly found an increased proportion of CD11c+CD163- pro-inflammatory peripheral monocytes in hypertensive subjects with VC compared to those without VC by flow cytometric analysis. Primary cultured macrophages from hypertensive subjects with VC also showed altered expression profile of inflammatory factors and higher serum OPN level. Exogenous OPN promoted the differentiation of peripheral monocytes into an alternative, anti-inflammatory phenotype, and inhibited macrophage-to-osteoclast differentiation from these VC patients. In addition, calcified vessels showed increased osteoclasts accumulation accompanied with decreased macrophages infiltration in the of hypertensive subjects. Taken together, these demonstrated that OPN exerts an important role in the monocytes/macrophage phenotypic differentiation from hypertensive patients with VC, which includes reducing inflammatory factor expression and attenuating osteoclast formation.

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. FASEB J. 31, 1120–1129 (2017). www.fasebj.org

Deficiency of Complement C3a and C5a Receptors Prevents Angiotensin II–Induced Hypertension via Regulatory T CellsNovelty and Significance

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.

9A.03: OSTEOPONTIN AND OSTEOPROTEGERIN ACTIVATE MONOCYTES INTO ANTI-INFLAMMATORY PROPERTIES IN THE PATIENTS WITH HYPERTENSION-RELATED VASCULAR CALCIFICATION.

Objective: Monocytes/macrophages are believed to play roles in vascular calcification(VC). Here, we analyzed whether osteopontin(OPN) and osteoprotegerin(OPG) might exert effects by promoting macrophage polarization into an anti-inflammatory phenotype in the patients with hypertension(HT)-related VC. Figure. No caption available. Design and method: In this study, 412 HT patients with or without VC were identified by using artery electronic calculators tomography(fig 1). Histological analysis was performed in the samples of aortic blood vessel from calcified vessels. Human peripheral blood CD14+ monocytes including M1∼like CD11c+ and M2∼like CD163+ cells were analysised by flow cytometery. The effects on M1 and M2 macrophages corrlated with cytokines and chemokines were assessed by qPCR. Results: We show that in HT patients,VC was correlated with higher systolic pressure,the higher incidence and more intima-media thickness of the plaque of carotid artery and was associated with arterial stiffness(including higher carotid-femoral pulse wave velocity, aortic systolic pressure, augment pressure, augment index(P < 0.05)). Furthermore, The phenotype of M1∼like monocyte/macrophages was significantly increasesd in HT patients with VC (P < 0.05)(Fig 3). Although both Serum OPN and OPG levels increased in HT patients with VC, they significantly upregulated anti-inflammatory M2 macrophages marks (P < 0.05) and only OPN downregulated pro-inflammatory M1 macrophages marks. Conclusions: The phenotype of M1 macrophages and M2 macrophages is promoted by VC(fig 2). The ability of OPN and OPG to promote differentiation of macrophages into an alternative, anti-inflammatory phenotype may explain their protective effects in VC of HT patients. These data provide novel insight into the link between inflammtion and VC diseases.

Loss of osteoglycin promotes angiogenesis in limb ischaemia mouse models via modulation of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 signalling pathway

Objective Osteoglycin (OGN) has been noted for its implication in cardiovascular disease in recent studies. However, the relationship between OGN and angiogenesis remains unknown. Therefore, we aimed to investigate the effect of OGN on ischaemia-induced angiogenesis and to address the underlying mechanisms. Methods and results The expression of OGN was decreased in a limb ischaemia mouse model. OGN knockout (KO) mice were used to further understand the role of OGN after ischaemia. The perfusion recovery rate after femoral artery ligation was higher in OGN KO mice than in wild-type (WT) mice. The capillary density in the gastrocnemius muscle of the ischaemic limb was also higher in OGN KO mice. Moreover, ex vivo aortic ring explants from OGN KO mice exhibited stronger angiogenic sprouting than those from WT mice. In human umbilical vein endothelial cells (HUVECs), OGN knockdown enhanced endothelial cell (EC) activation, including tube formation, proliferation, and migration. In contrast, OGN overexpression inhibited HUVEC activation. Mechanistic studies revealed that OGN associates with vascular endothelial growth factor receptor 2 (VEGFR2) and negatively regulates the interaction of vascular endothelial growth factor (VEGF) and VEGFR2, thereby negatively modulating the activation of VEGFR2 and its downstream signalling pathways. Consistently, the pro-angiogenic effect of OGN KO was abrogated by VEGFR2 inhibition, supporting the critical role of VEGFR2 signalling in OGN-mediated regulation of angiogenic function. Conclusions OGN plays a critical role in negatively regulating ischaemia-induced angiogenesis by inhibiting VEGF–VEGFR2 signalling and thereby attenuating EC tube formation, proliferation, and migration. Thus, OGN may be a novel therapeutic target for ischaemic vascular diseases.

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.

OS 23-02 THE ROLE OF COMPLEMENT C5a-MEDIATED PLACENTAL DYSFUNCTION IN THE ONSET OF PREECLAMPSIA.

Objective: Women with preeclampsia (PE) display excessive activation of complement system. However, little is known about the detailed cellular and molecular mechanism of complement activation in the development of PE. Here, we hypothesized that whether complement C5a contributes to the onset of PE through its effect on trophoblasts. Design and Method: In this study, 23 women with established PE and 32 normotensive women were recruited. At entry, peripheral and central blood pressure and pulse wave velocity (PWV) were performed. Immunofluorescence and quantitative real-time PCR were performed to identify the expression of C5a in the placenta. Transwell and matrigel assay was conducted to assess the effect of C5a on invasion and angiogenesis of human trophoblast cell lines. The serum level of C5a was measured by ELISA. Results: We detected an elevated C5a deposition in the placenta of patients with PE and C5aR was found highly expressed in syncytiotrophoblasts. In cultured trophoblast cell lines, C5aR agonist peptide inhibited the migration and angiogenesis of trophoblasts. C5aR agonist peptide stimulation also resulted in increased anti-angiogenic factors but decreased pro-angiogenic factors. In maternal circulation, the concentration of C5a was higher in women with PE compared to normotensive women (99.75 ± 29.27 ng/ml vs 76.35 ± 16.97 ng/ml mean ± SD P < 0.01). And meanwhile C5a has positive correlation with systolic blood pressure (r = 0.371, P < 0.01) and diastolic blood pressure (r = 0.343, P < 0.05). Women with PE displayed poor arterial function, which was also positively correlated with C5a level. Conclusions: Our data suggest that C5a contributed to the dysfunction of placenta by inhibition of migration and angiogenesis of trophoblast cells via C5aR in parallel with increase of blood pressure and arterial stiffness. The results indicate that C5a may have a novel role as a mediator of pathogenesis of PE, which could potentially result in gestational vascular dysfunction.

[BP.05.01] THE CLINICAL CHARACTERISTICS OF PATIENTS WITH PRIMARY ALDOSTERONISM COMPLICATED WITH OBSTRUCTIVE SLEEP APNEA - HYPOPNEA SYNDROME

Objective: This study aims to compare the differences of aldosterone, renin level and target organ damage in patients with primary aldosteronism complicated with or without OSAHS. Design and method: This study retrospectively analyzed patients who were diagnosed with PA in the Department of Hypertension, Ruijin Hospital during the period of 2010.1–2015.11. The cut-off value of plasma aldosterone to renin ratio (ARR) greater than 240 [ng / dl] / [ng / ml / h] was used to screen PA candidates, and plasma aldosterone greater than 60 ng / ml after saline infusion test was used as e PA confirmation criteria. According to the polysomnography, OSAHS was diagnosed with symptoms of apnea hypopnea index (AHI) > = 5 times / hour accompanied by nocturnal snoring or wakefulness or daytime sleepiness. Results: This study included 677 patients with PA, among them, 68 patients complicated with OSAHS. The prevalence of PA complicated OSAHS is 10.04%. Our study shows that PA with OSAHS group had significantly higher diastolic blood pressure (DBP), body mass index (BMI), triglyceride and low density lipoprotein level, left ventricular end-diastolic diameter and carotid intima-media thickness, and lower estimated glomerular filtration rate (eGFR) than those of patients without OSAHS group. In addition, the supine plasma renin activity of patients with PA and OSAHS was significantly higher, but plasma aldosterone level and ARR were significantly lower than those without OSAHS. When matched with age, sex, duration of hypertension and BMI, these two groups had no significant differences on plasma and urinary aldosterone levels, but the group with PA and OSAHS still had higher supine plasma renin activity and lower ARR and eGFR than the group without OSAHS. Furthermore, we found the lowest nocturnal oxyhemoglobin saturation was negatively correlated with BMI in OSAHS group(r = -0.318, P = 0.018). Conclusions: Patients with both PA and OSAHS had higher BMI, DBP, renin level and lower ARR and eGFR than those of patients without OSAHS. The difference of eGFR remained even after matching with age, BMI and duration of hypertension. OSAHS screening is therefore important in patients with PA. Figure. No caption available.

OS 35-06 COMPARISON OF N-TERMINAL PRO-B-TYPE NATRIURETIC PEPTIDE LEVEL IN PRIMARY ALDOSTERONISM AND ESSENTIAL HYPERTENSION.

Objective: Primary aldosteronism (PA) represents the most common cause of secondary hypertension. A higher risk of cardiovascular events has been reported in patients with PA than in otherwise similar patients with essential hypertension (EH). So far, only a few studies investigated the levels of plasma N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels in PA patients compared to EH patients. Design and Method: We studied 385 consecutive patients with PA and 385 with EH individually matched for age, gender, body mass index (BMI), blood pressure values and duration of hypertension. Fasting blood samples were obtained from all patients in our institution. Plasma levels of NT-proBNP and other clinical medical data were measured. Results: Two groups were similar in age, gender, BMI, office blood pressure and duration of hypertension. However, higher log-NT-proBNP level (1.74 ± 0.41 versus 1.50 ± 0.46, P < 0.001), left ventricular mass index (113 ± 25 versus 102 ± 26 g/m2, P < 0.001), night-time systolic blood pressure (SBP) (130 ± 16 versus 127 ± 17 mmHg, P = 0.01) and night-time diastolic blood pressure (DBP) (82 ± 10 versus 79 ± 11 mmHg, P < 0.01) were found in patients with PA compared with those with EH. The night-time BP decline was significantly attenuated in PA group. Univariate linear regression analysis showed that office systolic blood pressure (SBP) (r = 0.221, p < 0.001), 24 h mean SBP (r = 0.215, p < 0.001), plasma aldosterone levels were positively correlated with log-NT-proBNP levels. Conclusions: In this study, patients with PA show higher NT-proBNP levels and night-time blood pressure than those with EH. We hypothesize that these changes may play a role for the increased risk of future cardiovascular events in PA patients.