Brown Adipocyte ADRB3 Mediates Cardioprotection via Suppressing Exosomal iNOS.

BACKGROUND: The ADRB3 (ß3-adrenergic receptors), which is predominantly expressed in brown adipose tissue (BAT), can activate BAT and improve metabolic health. Previous studies indicate that the endocrine function of BAT is associated with cardiac homeostasis and diseases. Here, we investigate the role of ADRB3 activation-mediated BAT function in cardiac remodeling. METHODS: BKO (brown adipocyte-specific ADRB3 knockout) and littermate control mice were subjected to Ang II (angiotensin II) for 28 days. Exosomes from ADRB3 antagonist SR59230A (SR-exo) or agonist mirabegron (MR-exo) treated brown adipocytes were intravenously injected to Ang II-infused mice. RESULTS: BKO markedly accelerated cardiac hypertrophy and fibrosis compared with control mice after Ang II infusion. In vitro, ADRB3 KO rather than control brown adipocytes aggravated expression of fibrotic genes in cardiac fibroblasts, and this difference was not detected after exosome inhibitor treatment. Consistently, BKO brown adipocyte-derived exosomes accelerated Ang II-induced cardiac fibroblast dysfunction compared with control exosomes. Furthermore, SR-exo significantly aggravated Ang II-induced cardiac remodeling, whereas MR-exo attenuated cardiac dysfunction. Mechanistically, ADRB3 KO or SR59230A treatment in brown adipocytes resulted an increase of iNOS (inducible nitric oxide synthase) in exosomes. Knockdown of iNOS in brown adipocytes reversed SR-exo-aggravated cardiac remodeling. CONCLUSIONS: Our data illustrated a new endocrine pattern of BAT in regulating cardiac remodeling, suggesting that activation of ADRB3 in brown adipocytes offers cardiac protection through suppressing exosomal iNOS.

A2AR-mediated lymphangiogenesis via VEGFR2 signaling prevents salt-sensitive hypertension.

AIMS: Excess dietary sodium intake and retention lead to hypertension. Impaired dermal lymphangiogenesis and lymphatic dysfunction-mediated sodium and fluid imbalance are pathological mechanisms. The adenosine A2A receptor (A2AR) is expressed in lymphatic endothelial cells (LECs), while the roles and mechanisms of LEC-A2AR in skin lymphangiogenesis during salt-induced hypertension are not clear. METHODS AND RESULTS: The expression of LEC-A2AR correlated with lymphatic vessel density in both high-salt diet (HSD)-induced hypertensive mice and hypertensive patients. Lymphatic endothelial cell-specific A2AR knockout mice fed HSD exhibited 17 ± 2% increase in blood pressure and 17 ± 3% increase in Na+ content associated with decreased lymphatic density (-19 ± 2%) compared with HSD-WT mice. A2AR activation by agonist CGS21680 increased lymphatic capillary density and decreased blood pressure in HSD-WT mice. Furthermore, this A2AR agonist activated MSK1 directly to promote VEGFR2 activation and endocytosis independently of VEGF as assessed by phosphoprotein profiling and immunoprecipitation assays in LECs. VEGFR2 kinase activity inhibitor fruquintinib or VEGFR2 knockout in LECs but not VEGF-neutralizing antibody bevacizumab suppressed A2AR activation-mediated decrease in blood pressure. Immunostaining revealed phosphorylated VEGFR2 and MSK1 expression in the LECs were positively correlated with skin lymphatic vessel density and A2AR level in hypertensive patients. CONCLUSION: The study highlights a novel A2AR-mediated VEGF-independent activation of VEGFR2 signaling in dermal lymphangiogenesis and sodium balance, which might be a potential therapeutic target in salt-sensitive hypertension.

Jatrorrhizine Improves Endothelial Function in Diabetes and Obesity through Suppression of Endoplasmic Reticulum Stress.

Jatrorrhizine (JAT) is one of the major bioactive protoberberine alkaloids found in rhizoma coptidis, which has hypoglycemic and hypolipidemic potential. This study aimed to evaluate the vasoprotective effects of JAT in diabetes and obesity and the underlying mechanism involved. Mouse aortas, carotid arteries and human umbilical cord vein endothelial cells (HUVECs) were treated with risk factors (high glucose or tunicamycin) with and without JAT ex vivo and in vitro. Furthermore, aortas were obtained from mice with chronic treatment: (1) control; (2) diet-induced obese (DIO) mice fed a high-fat diet (45% kcal% fat) for 15 weeks; and (3) DIO mice orally administered JAT at 50 mg/kg/day for the last 5 weeks. High glucose or endoplasmic reticulum (ER) stress inducer tunicamycin impaired acetylcholine-induced endothelium-dependent relaxations (EDRs) in mouse aortas, induced oxidative stress in carotid arteries and HUVECs, downregulated phosphorylations of Akt at Ser473 and eNOS at Ser1177 and enhanced ER stress in mouse aortas and HUVECs, and these impairments were reversed by cotreatment with JAT. JAT increased NO release in high-glucose-treated mouse aortas and HUVECs. In addition, chronic JAT treatment restored endothelial function with EDRs comparable to the control, increased Akt/eNOS phosphorylation, and attenuated ER stress and oxidative stress in aortas from DIO mice. Blood pressure, glucose sensitivity, fatty liver and its morphological change, as well as plasma levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and plasma lipid profile, were also normalized by JAT treatment. Collectively, our data may be the first to reveal the vasoprotective effect of JAT that ameliorates endothelial dysfunction in diabetes and obesity through enhancement of the Akt/eNOS pathway and NO bioavailability, as well as suppression of ER stress and oxidative stress.

Adenosine A2A receptor activation prevents DOCA-salt induced hypertensive cardiac remodeling via iBAT.

Hypertensive cardiac remodeling is a constellation of abnormalities that includes cardiomyocyte hypertrophy and death and tissue fibrosis. Adenosine is a long-known vasodilator, through interacting with its four cell surface receptor subtypes in cardiovascular system. However, it is unclear that whether adenosine A2A receptor (A2AR) activation is involved in the cardiac remodeling in hypertension. WT mice were utilized to induce DOCA-salt sensitive hypertension and received A2AR agonist CGS21680 or antagonist KW6002 treatment. Cardiac functional phenotyping measurement by echocardiography showed that CGS21680 improved cardiac dysfunction in DOCA-salt mice. Moreover, CGS21680 reduced cardiomyocyte hypertrophy, cardiac inflammation and fibrosis. However, iBAT depletion surgery induces dramatic cardiac remodeling in DOCA-salt mice, and the protective function of CGS21680 was blocked without intact iBAT. Mechanistically, A2AR agonist CGS21680 increased iBAT-derived fibroblast growth factor 21 (FGF21). Our data suggest that activation of A2AR could be a potential therapeutic strategy in preventing heart damage in hypertension.

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.

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.

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.

Loss of miR-146b-3p Inhibits Perivascular Adipocyte Browning with Cold Exposure During Aging.

PURPOSE: Pathological changes of the perivascular adipose tissue (PVAT) are directly associated with increased risk of age-related vascular diseases. MicroRNAs regulate adipocyte biological functions including adipogenic differentiation and white adipocyte browning. The present study aims to determine whether miR-146b-3p is involved in the regulation of perivascular adipocyte browning during aging. METHODS: We utilized a cold-induced animal model to investigate the effect of aging on perivascular adipocyte browning. We also detected the miR-146b-3p expression in the PVAT of young or old mice after cold stimulus. We further investigated the role of miR-146b-3p in regulating perivascular adipocyte browning in vitro and in vivo via administrating miRNA mimics or inhibitors. RESULTS: Old mice showed decrease of perivascular adipocyte browning and downregulation of miR-146b-3p expression in the PVAT after cold stimulus. Oil red O staining and qPCR indicated that aging perturbed preadipocyte to brown adipocyte differentiation, and expression of miR-146b-3p gradually increased during differentiation. MiR-146b-3p inhibitors blocked brown adipocyte differentiation in young preadipocytes, whereas miR-146b-3p mimics rescued the differentiation of the old preadipocytes. Finally, miR-146b-3p knocks down inhibited perivascular adipocyte browning in young mice after cold stimulus. CONCLUSION: Aging inhibits perivascular adipocyte browning, and loss of miR-146b-3p is a potential regulator for this process.

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.

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.

The Characteristics of Macrophage Heterogeneity in Atherosclerotic Aortas.

Macrophage is the main effector cell during atherosclerosis. We applied single-cell RNA sequencing (scRNA) data to investigate the role of macrophage subsets in atherosclerosis. Monocyte and macrophage clusters were divided into 6 subclusters. Each subcluster's markers were calculated and validated by immunofluorescence. Elevated macrophage subclusters in the WD group were subject to enrichment pathway analysis and exhibited different phenotypes. Pseudotime analysis shows the subclusters originate from monocytes. We cultured bone marrow-derived macrophages with CSF-1 and ox-LDL to simulate an atherosclerotic-like environment and detected the transformation of subclusters. Macrophage-Vegfa and Macrophage-C1qb increased in the WD group. Macrophage-Vegfa acquires the characteristics of phagocytosis and immune response, while Macrophage-C1qb is not involved in lipid metabolism. The two subclusters are both enriched in cell movement and migration pathways. Experimental verification proved Monocyte-Ly6C evolved into Macrophage-Vegfa and Macrophage-C1qb during atherosclerosis progression.

Ginsenoside Rk1 improves endothelial function in diabetes through activating peroxisome proliferator-activated receptors.

Ginsenoside Rk1, one kind of ginsenoside, is a minor ginsenoside found in Panax ginseng and used as traditional Chinese medicine for centuries. It exhibits anti-tumor and anti-aggregation effects. However, little research has been done on its effect on endothelial function. This study investigated whether ginsenoside Rk1 improved endothelial dysfunction in diabetes and the underlying mechanisms in vivo and in vitro. Male C57BL/6 mice were fed with a 12 week high-fat diet (60% kcal % fat), whereas treatment groups were orally administered with ginsenoside Rk1 (10 and 20 mg per kg per day) in the last 4 weeks. Aortas isolated from C57BL/6 mice were induced by high glucose (HG; 30 mM) and co-treated with or without ginsenoside Rk1 (1 and 10 µM) for 48 h ex vivo. Moreover, primary rat aortic endothelial cells (RAECs) were cultured and stimulated by HG (44 mM) to mimic hyperglycemia, with or without the co-treatment of ginsenoside Rk1 (10 µM) for 48 h. Endothelium-dependent relaxations of mouse aortas were damaged with elevated oxidative stress and downregulation of three isoforms of peroxisome proliferator-activated receptors (PPARs), PPAR-α, PPAR-ß/δ, and PPAR-γ, as well as endothelial nitric oxide synthase (eNOS) phosphorylation due to HG or high-fat diet stimulation, which also existed in RAECs. However, after the treatment with ginsenoside Rk1, these impairments were all ameliorated significantly. Moreover, the vaso-protective and anti-oxidative effects of ginsenoside Rk1 were abolished by PPAR antagonists (GSK0660, GW9662 or GW6471). In conclusion, this study reveals that ginsenoside Rk1 ameliorates endothelial dysfunction and suppresses oxidative stress in diabetic vasculature through activating the PPAR/eNOS pathway.

Improvement of MASLD and MASH by suppression of hepatic N-acetyltransferase 10.

OBJECTIVE: Metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH) are characterized by excessive triglyceride accumulation in the liver. However, due to an incomplete understanding of its pathogenesis, more efforts are needed to identify specific and effective treatments. N4-acetylcytidine (ac4C) is a newly discovered RNA modification to regulate mRNA. N-acetyltransferase 10 (NAT10) has not been fully explored in MASLD and MASH. METHODS: The clinical relevance of NAT10 was evaluated based on its expression in various mouse and human models of MASLD and MASH. Acetylated RNA immunoprecipitation sequencing and mRNA stability assays were used to explore the role of NAT10 in regulating ac4C modification and expression of target genes. Genetically engineered mice were employed to investigate the role of NAT10 in MASLD and MASH progression. RESULTS: Hepatic NAT10 expression was significantly increased in multiple mice and humans of MASLD and MASH. Genetic knockout of NAT10 protected mice from diet-induced hepatic steatosis and steatohepatitis, whereas overexpression of NAT10 exacerbated high-fat-diet-induced liver steatosis. Mechanistically, NAT10 binds to Srebp-1c mRNA, promoting its stability and expression, thereby upregulating lipogenic enzymes. Treatment with Remodelin, a NAT10-specific inhibitor, effectively ameliorates liver steatosis and dyslipidemia in a preclinical mouse model. CONCLUSIONS: Our findings indicate that NAT10 could regulate lipid metabolism in MASLD and MASH by stabilizing Srebp-1c mRNA and upregulating lipogenic enzymes. This study highlights the role of NAT10 and RNA acetylation in the pathogenesis of MASLD and MASH. Thus, our findings suggest a promising new therapeutic approach, such as the use of NAT10 inhibitor, for treating metabolic liver disease.

ALKBH5-mediated m6A modification of IL-11 drives macrophage-to-myofibroblast transition and pathological cardiac fibrosis in mice.

Cardiac macrophage contributes to the development of cardiac fibrosis, but factors that regulate cardiac macrophages transition and activation during this process remains elusive. Here we show, by single-cell transcriptomics, lineage tracing and parabiosis, that cardiac macrophages from circulating monocytes preferentially commit to macrophage-to-myofibroblast transition (MMT) under angiotensin II (Ang II)-induced hypertension, with accompanying increased expression of the RNA N6-methyladenosine demethylases, ALKBH5. Meanwhile, macrophage-specific knockout of ALKBH5 inhibits Ang II-induced MMT, and subsequently ameliorates cardiac fibrosis and dysfunction. Mechanistically, RNA immunoprecipitation sequencing identifies interlukin-11 (IL-11) mRNA as a target for ALKBH5-mediated m6A demethylation, leading to increased IL-11 mRNA stability and protein levels. By contrast, overexpression of IL11 in circulating macrophages reverses the phenotype in ALKBH5-deficient mice and macrophage. Lastly, targeted delivery of ALKBH5 or IL-11 receptor α (IL11RA1) siRNA to monocytes/macrophages attenuates MMT and cardiac fibrosis under hypertensive stress. Our results thus suggest that the ALKBH5/IL-11/IL11RA1/MMT axis alters cardiac macrophage and contributes to hypertensive cardiac fibrosis and dysfunction in mice, and thereby identify potential targets for cardiac fibrosis therapy in patients.

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.

Isolation and Characterization of Brown Adipose Tissue T Cells.

Brown adipose tissue (BAT) is a specialized fat depot that can dissipate energy through uncoupled respiration and thermogenesis. Various immune cells such as macrophages, eosinophils, type 2 innate lymphoid cells, and T lymphocytes were recently found to have an unexpected involvement in controlling the thermogenic activity of brown adipose tissue. Here, we describe a protocol for isolation and characterization of T cells from brown adipose tissue.

An IL-6/STAT3/MR/FGF21 axis mediates heart-liver cross-talk after myocardial infarction.

The liver plays a protective role in myocardial infarction (MI). However, very little is known about the mechanisms. Here, we identify mineralocorticoid receptor (MR) as a pivotal nexus that conveys communications between the liver and the heart during MI. Hepatocyte MR deficiency and MR antagonist spironolactone both improve cardiac repair after MI through regulation on hepatic fibroblast growth factor 21 (FGF21), illustrating an MR/FGF21 axis that underlies the liver-to-heart protection against MI. In addition, an upstreaming acute interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) pathway transmits the heart-to-liver signal to suppress MR expression after MI. Hepatocyte Il6 receptor deficiency and Stat3 deficiency both aggravate cardiac injury through their regulation on the MR/FGF21 axis. Therefore, we have unveiled an IL-6/STAT3/MR/FGF21 signaling axis that mediates heart-liver cross-talk during MI. Targeting the signaling axis and the cross-talk could provide new strategies to treat MI and heart failure.

Leonurine Attenuates Obesity-Related Vascular Dysfunction and Inflammation.

Oxidative stress in adipose tissue is a crucial pathogenic mechanism of obesity-associated cardiovascular diseases. Chronic low-grade inflammation caused by obesity increases ROS production and dysregulation of adipocytokines. Leonurine (LEO) is an active alkaloid extracted from Herba Leonuri and plays a protective role in the cardiovascular system. The present study tested whether LEO alleviates inflammation and oxidative stress, and improves vascular function in an obese mouse model. Here, we found that obesity leads to inflammation and oxidative stress in epididymal white adipose tissue (EWAT), as well as vascular dysfunction. LEO significantly improved inflammation and oxidative stress both in vivo and in vitro. Obesity-induced vascular dysfunction was also improved by LEO as evidenced by the ameliorated vascular tone and decreased mesenteric artery fibrosis. Using mass spectrometry, we identified YTHDF1 as the direct target of LEO. Taken together, we demonstrated that LEO improves oxidative stress and vascular remodeling induced by obesity and targets YTHDF1, raising the possibility of LEO treating other obesity-related metabolic syndromes.

Perivascular adipose tissue-derived complement 3 is required for adventitial fibroblast functions and adventitial remodeling in deoxycorticosterone acetate-salt hypertensive rats.

OBJECTIVE: To examine the role of perivascular adipose tissue (PVAT)-derived factors in the regulation of adventitial fibroblast (AF) function in vitro and in vivo. METHODS AND RESULTS: PVAT is an active component of blood vessels. Bioactive substances released from PVAT play regulatory roles in vascular function. However, their effects on vascular AFs remain unclear. PVAT-conditioned medium stimulated AF migration using a transwell technique, and differentiation was evaluated by α-smooth muscle-actin induction. We identified the secretome of PVAT by liquid chromatography-tandem mass spectrometry. One of the major secretory proteins in PVAT is complement 3 (C3). The C3 antagonist and neutralizing antibody attenuated PVAT-conditioned medium-induced AF migration and differentiation. Similar to PVAT-conditioned medium, C3 recombinant protein stimulated AF migration and differentiation. We demonstrated that the effects of PVAT-derived C3 were mediated by the c-Jun N-terminal kinase pathway. Moreover, we found morphological changes in perivascular adipocytes and increased expression of C3 in PVAT that was tightly associated with adventitial thickening and myofibroblast clustering around PVAT in deoxycorticosterone acetate-salt hypertensive rats. CONCLUSIONS: PVAT-derived C3 stimulated AF migration and differentiation via the c-Jun N-terminal kinase pathway. PVAT-derived C3 may contribute to adventitial remodeling in a deoxycorticosterone acetate-salt hypertensive model.