Cheng-Chao Ruan

Activating transcription factor 3 SUMOylation is involved in angiotensin II-induced endothelial cell inflammation and dysfunction

Activating transcription factor 3 (ATF3) is an adaptive-response protein induced by various environmental stresses and is implicated in the pathogenesis of many disease states. However, the role of ATF3 SUMOylation in hypertension-induced vascular injury remains poorly understood. Here we investigated the function of ATF3 SUMOylation in vascular endothelial cells (ECs). The expression of ATF3 and small ubiquitin-like modifier 1 (SUMO1) was increased in angiotensin II (Ang II)-induced human umbilical vein endothelial cells (HUVECs). Microscopic analyses further revealed that the expression of ATF3 and SUMO1 is upregulated and colocalized in the endothelium of thoracic aortas from Ang II-induced hypertensive mice. However, Ang II-induced upregulation of ATF3 and SUMO1 in vitro and in vivo was blocked by Ang II type I receptor antagonist olmesartan. Moreover, Ang II induced ATF3 SUMOylation at lysine 42, which is SUMO1 dependent. ATF3 SUMOylation attenuated ATF3 ubiquitination and in turn promoted ATF3 protein stability. ATF3 or SUMO1 knockdown inhibited Ang II-induced expression of inflammatory molecules such as tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8. Wild type ATF3 but not ATF3-K42R (SUMOylation defective mutant) reduced the production of nitric oxide (NO), a key indicator of EC function. Consistently, ginkgolic acid, an inhibitor of SUMOylation, increased NO production in HUVECs and significantly improved vasodilatation of aorta from Ang II-induced hypertensive mice. Our findings demonstrated that ATF3 SUMOylation is involved in Ang II-induced EC inflammation and dysfunction in vitro and in vivo through inhibiting ATF3 ubiquitination and increasing ATF3 protein stability.

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.

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-β (TGF-β)/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.

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.

Decrease of Perivascular Adipose Tissue Browning Is Associated With Vascular Dysfunction in Spontaneous Hypertensive Rats During Aging

Functional perivascular adipose tissue (PVAT) is necessary to maintain vascular physiology through both mechanical support and endocrine or paracrine ways. PVAT shows a brown adipose tissue (BAT)-like feature and the browning level of PVAT is dependent on the anatomic location and species. However, it is not clear whether PVAT browning is involved in the vascular tone regulation in spontaneously hypertensive rats (SHRs). In the present study, we aimed to illustrate the effect of aging on PVAT browning and subsequent vasomotor reaction in SHRs. Herein we utilized histological staining and western blot to detect the characteristics of thoracic PVAT (tPVAT) in 8-week-old and 16-week-old SHR and Wistar-Kyoto (WKY) rats. We also detected vascular reactivity analysis to determine the effect of tPVAT on vasomotor reaction during aging. The results showed that tPVAT had a similar phenotype to BAT, including smaller adipocyte size and positive uncoupling protein-1 (UCP1) staining. Interestingly, the tPVAT of 8-week-old SHR showed increased BAT phenotypic marker expression compared to WKY, whereas the browning level of tPVAT had a more dramatic decrease from 8 to 16 weeks of age in SHR than age-matched WKY rats. The vasodilation effect of tPVAT on aortas had no significant difference in 8-week-old WKY and SHR, whereas this effect is obviously decreased in 16-week-old SHR compared to WKY. In contrast, tPVAT showed a similar vasoconstriction effect in 8- or 16-week-old WKY and SHR rats. Moreover, we identified an important vasodilator adenosine, which regulates adipocyte browning and may be a potential PVAT-derived relaxing factor. Adenosine is dramatically decreased from 8 to 16 weeks of age in the tPVAT of SHR. In summary, aging is associated with a decrease of tPVAT browning and adenosine production in SHR rats. These may result in attenuated vasodilation effect of the tPVAT in SHR during aging.

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.

Beta3 adrenergic receptor is involved in vascular injury in deoxycorticosterone acetate‐salt hypertensive mice

Beta3 adrenergic receptor (ADRB3) mediates vessel relaxation in the endothelium while it modulates lipolysis in the adipose tissue. However, the function and regulation mechanism of ADRB3 in the perivascular adipose tissue (PVAT), especially in hypertension, is still unclear. We show that ADRB3 protein is upregulated in the PVAT of deoxycorticosterone acetate‐salt (DOCA‐salt) hypertensive mice, with the characteristics of PVAT browning and increased uncoupling protein 1 (UCP1) expression. Inhibition of ADRB3 with selective antagonist SR59230A caused serious vascular injury in vivo, even though UCP1 expression was downregulated. ADRB3 protein was regulated by let‐7b, which was decreased in the PVAT of the DOCA‐salt group. These data reveal that ADRB3 in PVAT contributes to vascular function in the progression of hypertension.

Perivascular Adipose Tissue-Derived PDGF-D Contributes to Aortic Aneurysm Formation during Obesity

Obesity increases the risk of vascular diseases, including aortic aneurysm (AA). Perivascular adipose tissue (PVAT) surrounding arteries are altered during obesity. However, the underlying mechanism of adipose tissue, especially PVAT, in the pathogenesis of AA is still unclear. Here we showed that angiotensin II (AngII) infusion increases the incidence of AA in leptin-deficient obese mice (ob/ob) and high-fat diet–induced obese mice with adventitial inflammation. Furthermore, transcriptome analysis revealed that platelet-derived growth factor-D (PDGF-D) was highly expressed in the PVAT of ob/ob mice. Therefore, we hypothesized that PDGF-D mediates adventitial inflammation, which provides a direct link between PVAT dysfunction and AA formation in AngII-infused obese mice. We found that PDGF-D promotes the proliferation, migration, and inflammatory factors expression in cultured adventitial fibroblasts. In addition, the inhibition of PDGF-D function significantly reduced the incidence of AA in AngII-infused obese mice. More importantly, adipocyte-specific PDGF-D transgenic mice are more susceptible to AA formation after AngII infusion accompanied by exaggerated adventitial inflammatory and fibrotic responses. Collectively, our findings reveal a notable role of PDGF-D in the AA formation during obesity, and modulation of this cytokine might be an exploitable treatment strategy for the condition.