Endothelial cell Orai1 is essential for endothelium-dependent contraction of mouse carotid arteries in normotensive and hypertensive mice.

Endothelium-dependent contraction (EDC) exists in blood vessels of normotensive animals, but is exaggerated in hypertension. An early signal in EDC is cytosolic Ca2+ rise in endothelial cells. In this study we investigated the functional role of Orai1, a major endothelial cell Ca2+ entry channel, in EDC. Hypertension model was established in WT mice by intake of L-NNA in the drinking water (0.5 g/L) for 4 weeks or osmotic pump delivery of Ang II (1.5 mg·kg-1·d-1) for 2 weeks. In TRPC5 KO mice, the concentration of L-NNA and Ang II were increased to 1 g/L or 2 mg·kg-1·d-1, respectively. Arterial segments were prepared from carotid arteries and aortas, and EDC was elicited by acetylcholine in the presence of Nω-nitro-L-arginine methyl ester. We showed that low concentration of acetylcholine (3-30 nM) initiated relaxation in phenylephrine-precontracted carotid arteries of both normotensive and hypertensive mice, while high concentration of acetylcholine (0.1-2 µM) induced contraction. Application of selective Orai1 inhibitors AnCoA4 (100 µM) or YM58483 (400 nM) had no effect on ACh-induced relaxation but markedly reduced acetylcholine-induced EDC. We found that EDC was increased in hypertensive mice compared with that of normotensive mice, which was associated with increased Orai1 expression in endothelial cells of hypertensive mice. Compared to TRPC5 and TRPV4, which were also involved in EDC, endothelial cell Orai1 had relatively greater contribution to EDC than either TRPC5 or TRPV4 alone. We identified COX-2, followed by PGF2α, PGD2 and PGE2 as the downstream signals of Orai1/TRPC5/TRPV4. In conclusion, Orai1 coordinates together with TRPC5 and TRPV4 in endothelial cells to regulate EDC responses. This study demonstrates a novel function of Orai1 in EDC in both normotensive and hypertensive mice, thus providing a general scheme about the control of EDC by Ca2+-permeable channels.

The role of KLF2 in regulating hepatic lipogenesis and blood cholesterol homeostasis via the SCAP/SREBP pathway.

Liver steatosis is a common metabolic disorder resulting from imbalanced lipid metabolism, which involves various processes such as de novo lipogenesis, fatty acid uptake, fatty acid oxidation, and VLDL secretion. In this study, we discovered that KLF2, a transcription factor, plays a crucial role in regulating lipid metabolism in the liver. Overexpression of KLF2 in the liver of db/db mice, C57BL/6J mice, and Cd36-/- mice fed on a normal diet resulted in increased lipid content in the liver. Additionally, transgenic mice (ALB-Klf2) that overexpressed Klf2 in the liver developed liver steatosis after being fed a normal diet. We found that KLF2 promotes lipogenesis by increasing the expression of SCAP, a chaperone that facilitates the activation of SREBP, the master transcription factor for lipogenic gene expression. Our mechanism studies revealed that KLF2 enhances lipogenesis in the liver by binding to the promoter of SCAP and increasing the expression of genes involved in fatty acid synthesis. Reduction of KLF2 expression led to a decrease in SCAP expression and a reduction in the expression of SREBP1 target genes involved in lipogenesis. Overexpression of KLF2 also increased the activation of SREBP2 and the mRNA levels of its downstream target SOAT1. In C57BL/6J mice fed a high-fat diet, overexpression of Klf2 increased blood VLDL secretion, while reducing its expression decreased blood cholesterol levels. Our study emphasizes the novelty that hepatic KLF2 plays a critical role in regulating lipid metabolism through the KLF2/SCAP/SREBPs pathway, which is essential for hepatic lipogenesis and maintaining blood cholesterol homeostasis.

AMPK-Dependent YAP Inhibition Mediates the Protective Effect of Metformin against Obesity-Associated Endothelial Dysfunction and Inflammation.

Hyperglycemia is a crucial risk factor for cardiovascular diseases. Chronic inflammation is a central characteristic of obesity, leading to many of its complications. Recent studies have shown that high glucose activates Yes-associated protein 1 (YAP) by suppressing AMPK activity in breast cancer cells. Metformin is a commonly prescribed anti-diabetic drug best known for its AMPK-activating effect. However, the role of YAP in the vasoprotective effect of metformin in diabetic endothelial cell dysfunction is still unknown. The present study aimed to investigate whether YAP activation plays a role in obesity-associated endothelial dysfunction and inflammation and examine whether the vasoprotective effect of metformin is related to YAP inhibition. Reanalysis of the clinical sequencing data revealed YAP signaling, and the YAP target genes CTGF and CYR61 were upregulated in aortic endothelial cells and retinal fibrovascular membranes from diabetic patients. YAP overexpression impaired endothelium-dependent relaxations (EDRs) in isolated mouse aortas and increased the expression of YAP target genes and inflammatory markers in human umbilical vein endothelial cells (HUVECs). High glucose-activated YAP in HUVECs and aortas was accompanied by increased production of oxygen-reactive species. AMPK inhibition was found to induce YAP activation, resulting in increased JNK activity. Metformin activated AMPK and promoted YAP phosphorylation, ultimately improving EDRs and suppressing the JNK activity. Targeting the AMPK-YAP-JNK axis could become a therapeutic strategy for alleviating vascular dysfunction in obesity and diabetes.

Induction of KLF2 by Exercise Activates eNOS to Improve Vasodilatation in Diabetic Mice.

Diabetic endothelial dysfunction associated with diminished endothelial nitric oxide (NO) synthase (eNOS) activity accelerates the development of atherosclerosis and cardiomyopathy. However, the approaches to restore eNOS activity and endothelial function in diabetes remain limited. The current study shows that enhanced expression of Krüppel-like factor 2 (KLF2), a shear stress-inducible transcription factor, effectively improves endothelial function through increasing NO bioavailability. KLF2 expression is suppressed in diabetic mouse aortic endothelium. Running exercise and simvastatin treatment induce endothelial KLF2 expression in db/db mice. Adenovirus-mediated endothelium-specific KLF2 overexpression enhances both endothelium-dependent relaxation and flow-mediated dilatation, while it attenuates oxidative stress in diabetic mouse arteries. KLF2 overexpression increases the phosphorylation of eNOS at serine 1177 and eNOS dimerization. RNA-sequencing analysis reveals that KLF2 transcriptionally upregulates genes that are enriched in the cyclic guanosine monophosphate-protein kinase G-signaling pathway, cAMP-signaling pathway, and insulin-signaling pathway, all of which are the upstream regulators of eNOS activity. Activation of the phosphoinositide 3-kinase-Akt pathway and Hsp90 contributes to KLF2-induced increase of eNOS activity. The present results suggest that approaches inducing KLF2 activation, such as physical exercise, are effective to restore eNOS activity against diabetic endothelial dysfunction. ARTICLE HIGHLIGHTS: Exercise and statins restore the endothelial expression of Krüppel-like factor 2 (KLF2), which is diminished in diabetic db/db mice. Endothelium-specific overexpression of KLF2 improves endothelium-dependent relaxation and flow-mediated dilation through increasing nitric oxide bioavailability. KLF2 promotes endothelial nitric oxide synthase (eNOS) coupling and phosphorylation in addition to its known role in eNOS transcription. KLF2 upregulates the expression of several panels of genes that regulate eNOS activity.

Thioridazine protects against disturbed flow-induced atherosclerosis by inhibiting RhoA/YAP-mediated endothelial inflammation.

Atherosclerotic diseases remain the leading cause of adult mortality and impose heavy burdens on health systems globally. Our previous study found that disturbed flow enhanced YAP activity to provoke endothelial activation and atherosclerosis, and targeting YAP alleviated endothelial inflammation and atherogenesis. Therefore, we established a luciferase reporter assay-based drug screening platform to seek out new YAP inhibitors for anti-atherosclerotic treatment. By screening the FDA-approved drug library, we identified that an anti-psychotic drug thioridazine markedly suppressed YAP activity in human endothelial cells. Thioridazine inhibited disturbed flow-induced endothelial inflammatory response in vivo and in vitro. We verified that the anti-inflammatory effects of thioridazine were mediated by inhibition of YAP. Thioridazine regulated YAP activity via restraining RhoA. Moreover, administration of thioridazine attenuated partial carotid ligation- and western diet-induced atherosclerosis in two mouse models. Overall, this work opens up the possibility of repurposing thioridazine for intervention of atherosclerotic diseases. This study also shed light on the underlying mechanisms that thioridazine inhibited endothelial activation and atherogenesis via repression of RhoA-YAP axis. As a new YAP inhibitor, thioridazine might need further investigation and development for the treatment of atherosclerotic diseases in clinical practice.

Smad4-mediated angiogenesis facilitates the beiging of white adipose tissue in mice.

Beige adipocytes are thermogenic with high expression of uncoupling protein 1 in the white adipose tissue (WAT), accompanied by angiogenesis. Previous studies showed that Smad4 is important for angiogenesis. Here we studied whether endothelial Smad4-mediated angiogenesis is involved in WAT beiging. Inducible knockout of endothelial cell (EC) selective Smad4 (Smad4 iEC-KO) was achieved by using the Smad4 Floxp/floxp and Tie2 CreERT2 mice. Beige fat induction achieved by cold or adrenergic agonist, and angiogenesis were attenuated in WAT of Smad4 iEC-KO mice, with the less proliferation of ECs and adipogenic precursors. RNA sequencing of human ECs showed that Smad4 is involved in angiogenesis-related pathways. Knockdown of SMAD4 attenuated the upregulation of VEGFA, PDGFA, and angiogenesis in vitro. Treatment of human ECs with palmitic acid-induced Smad1/5 phosphorylation and the upregulation of core endothelial genes. Our study shows that endothelial Smad4 is involved in WAT beiging through angiogenesis and the expansion of adipose precursors into beige adipocytes.

SOX4 is a novel phenotypic regulator of endothelial cells in atherosclerosis revealed by single-cell analysis.

INTRODUCTION: Atherosclerotic complications represent the leading cause of cardiovascular mortality globally. Dysfunction of endothelial cells (ECs) often initiates the pathological events in atherosclerosis. OBJECTIVES: In this study, we sought to investigate the transcriptional profile of atherosclerotic aortae, identify novel regulator in dysfunctional ECs and hence provide mechanistic insights into atherosclerotic progression. METHODS: We applied single-cell RNA sequencing (scRNA-seq) on aortic cells from Western diet-fed apolipoprotein E-deficient (ApoE-/-) mice to explore the transcriptional landscape and heterogeneity of dysfunctional ECs. In vivo validation of SOX4 upregulation in ECs were performed in atherosclerotic tissues, including mouse aortic tissues, human coronary arteries, and human renal arteries. Single-cell analysis on human aortic aneurysmal tissue was also performed. Downstream vascular abnormalities induced by EC-specific SOX4 overexpression, and upstream modulators of SOX4 were revealed by biochemical assays, immunostaining, and wire myography. Effects of shear stress on endothelial SOX4 expression was investigated by in vitro hemodynamic study. RESULTS: Among the compendium of aortic cells, mesenchymal markers in ECs were significantly enriched. Two EC subsets were subsequently distinguished, as the 'endothelial-like' and 'mesenchymal-like' subsets. Conventional assays consistently identified SOX4 as a novel atherosclerotic marker in mouse and different human arteries, additional to a cancer marker. EC-specific SOX4 overexpression promoted atherogenesis and endothelial-to-mesenchymal transition (EndoMT). Importantly, hyperlipidemia-associated cytokines and oscillatory blood flow upregulated, whereas the anti-diabetic drug metformin pharmacologically suppressed SOX4 level in ECs. CONCLUSION: Our study unravels SOX4 as a novel phenotypic regulator during endothelial dysfunction, which exacerbates atherogenesis. Our study also pinpoints hyperlipidemia-associated cytokines and oscillatory blood flow as endogenous SOX4 inducers, providing more therapeutic insights against atherosclerotic diseases.

Curaxin CBL0137 inhibits endothelial inflammation and atherogenesis via suppression of the Src-YAP signalling axis.

BACKGROUND AND PURPOSE: Atherosclerotic vascular disease is the leading cause of mortality and morbidity worldwide. Our previous study uncovered that endothelium-specific knockdown of YAP suppresses atherogenesis, suggesting that YAP is a promising therapeutic target against atherosclerotic vascular disease. We established a drug screening platform, which aimed to identify new YAP inhibitors for anti-atherosclerotic treatment. EXPERIMENTAL APPROACH: Drug screening was performed by a luciferase reporter gene assay. RNA sequencing was performed to acquire the transcriptomic profile of CBL0137-treated endothelial cells. We assessed and validated the inhibitory effect of CBL0137 on YAP activity and inflammatory response in HUVECs and HAECs. We evaluated the vasoprotective effect of CBL0137 in vivo against plaque formation in ApoE-/- mice, using both disturbed flow-induced and chronic western diet-induced atherosclerotic models. KEY RESULTS: We identified CBL0137 as a novel YAP inhibitor from an FDA drug library. CBL0137 inhibited YAP activity by restraining its phosphorylation at Y357. CBL0137 inhibited YAP activity to repress endothelial inflammation. Mechanistically, CBL0137 suppressed YAP phosphorylation at Y357 via the tyrosine-protein kinase Src. Furthermore, administration of CBL0137 ameliorated endothelial inflammation and the atherogenesis induced by disturbed flow and consumption of an atherogenic diet in ApoE-/- mice. CONCLUSION AND IMPLICATIONS: To our knowledge, this is the first study to identify CBL0137 as a novel YAP inhibitor. We have demonstrated that pharmacologically targeting YAP by CBL0137 inhibits atherogenesis. The present results suggest that CBL0137 holds promise as a new drug for the treatment of atherosclerotic vascular disease.

Endothelial UCP2 Is a Mechanosensitive Suppressor of Atherosclerosis.

BACKGROUND: Inflamed endothelial cells (ECs) trigger atherogenesis, especially at arterial regions experiencing disturbed blood flow. UCP2 (Uncoupling protein 2), a key mitochondrial antioxidant protein, improves endothelium-dependent relaxation in obese mice. However, whether UCP2 can be regulated by shear flow is unknown, and the role of endothelial UCP2 in regulating inflammation and atherosclerosis remains unclear. This study aims to investigate the mechanoregulation of UCP2 expression in ECs and the effect of UCP2 on endothelial inflammation and atherogenesis. METHODS: In vitro shear stress simulation system was used to investigate the regulation of UCP2 expression by shear flow. EC-specific Ucp2 knockout mice were used to investigate the role of UCP2 in flow-associated atherosclerosis. RESULTS: Shear stress experiments showed that KLF2 (Krüppel-like factor 2) mediates fluid shear stress-dependent regulation of UCP2 expression in human aortic and human umbilical vein ECs. Unidirectional shear stress, statins, and resveratrol upregulate whereas oscillatory shear stress and proinflammatory stimuli inhibit UCP2 expression through altered KLF2 expression. KLF2 directly binds to UCP2 promoter to upregulate its transcription in human umbilical vein ECs. UCP2 knockdown induced expression of genes involved in proinflammatory and profibrotic signaling, resulting in a proatherogenic endothelial phenotype. EC-specific Ucp2 deletion promotes atherogenesis and collagen production. Additionally, we found endothelial Ucp2 deficiency aggravates whereas adeno-associated virus-mediated EC-Ucp2 overexpression inhibits carotid atherosclerotic plaque formation in disturbed flow-enhanced atherosclerosis mouse model. RNA-sequencing analysis revealed FoxO1 (forkhead box protein O1) as the major proinflammatory transcriptional regulator activated by UCP2 knockdown, and FoxO1 inhibition reduced vascular inflammation and disturbed flow-enhanced atherosclerosis. We showed further that UCP2 level is critical for phosphorylation of AMPK (AMP-activated protein kinase), which is required for UCP2-induced inhibition of FoxO1. CONCLUSIONS: Altogether, our studies uncover that UCP2 is novel mechanosensitive gene under the control of fluid shear stress and KLF2 in ECs. UCP2 expression is critical for endothelial proinflammatory response and atherogenesis. Therapeutic strategies enhancing UCP2 level may have therapeutic potential against atherosclerosis.

Activation of AMPK/miR-181b Axis Alleviates Endothelial Dysfunction and Vascular Inflammation in Diabetic Mice.

Hyperglycemia in diabetes mellitus impairs endothelial function and disrupts microRNA (miRNA) profiles in vasculature, increasing the risk of diabetes-associated complications, including coronary artery disease, diabetic retinopathy, and diabetic nephropathy. miR-181b was previously reported to be an anti-inflammatory mediator in vasculature against atherosclerosis. The current study aimed to investigate whether miR-181b ameliorates diabetes-associated endothelial dysfunction, and to identify potential molecular mechanisms and upstream inducer of miR-181b. We found that miR-181b level was decreased in renal arteries of diabetic patients and in advanced glycation end products (AGEs)-treated renal arteries of non-diabetic patients. Transfection of miR-181b mimics improved endothelium-dependent vasodilation in aortas of high fat diet (HFD)/streptozotocin (STZ)-induced diabetic mice, accompanied by suppression of superoxide overproduction and vascular inflammation markers. AMPK activator-induced AMPK activation upregulated miR-181b level in human umbilical vein endothelial cells (HUVECs). Chronic exercise, potentially through increased blood flow, activated AMPK/miR-181b axis in aortas of diabetic mice. Exposure to laminar shear stress upregulated miR-181b expression in HUVECs. Overall, our findings highlight a critical role of AMPK/miR-181b axis and extend the benefits of chronic exercise in counteracting diabetes-associated endothelial dysfunction.

MicroRNA-29a attenuates CD8 T cell exhaustion and induces memory-like CD8 T cells during chronic infection.

CD8 T cells mediate protection against intracellular pathogens and tumors. However, persistent antigen during chronic infections or cancer leads to T cell exhaustion, suboptimal functionality, and reduced protective capacity. Despite considerable work interrogating the transcriptional regulation of exhausted CD8 T cells (TEX), the posttranscriptional control of TEX remains poorly understood. Here, we interrogated the role of microRNAs (miRs) in CD8 T cells responding to acutely resolved or chronic viral infection and identified miR-29a as a key regulator of TEX. Enforced expression of miR-29a improved CD8 T cell responses during chronic viral infection and antagonized exhaustion. miR-29a inhibited exhaustion-driving transcriptional pathways, including inflammatory and T cell receptor signaling, and regulated ribosomal biogenesis. As a result, miR-29a fostered a memory-like CD8 T cell differentiation state during chronic infection. Thus, we identify miR-29a as a key regulator of TEX and define mechanisms by which miR-29a can divert exhaustion toward a more beneficial memory-like CD8 T cell differentiation state.

Branched-chain amino acid supplementation impairs insulin sensitivity and promotes lipogenesis during exercise in diet-induced obese mice.

OBJECTIVE: Branched-chain amino acids (BCAAs) are popular dietary supplements for exercise. However, increased BCAA levels positively correlate with obesity and diabetes. The metabolic impact of BCAA supplementation on insulin sensitivity during exercise is less understood. METHODS: Male C57BL/6 mice were fed for 12 weeks with a high-fat diet, normal chow diet, or BCAA-restricted high-fat diet. They were subjected to running exercise with or without BCAA treatment for another 12 weeks. RESULTS: Exercise reduced body weight, improved insulin sensitivity, lowered BCAAs in plasma, and inhibited the upregulation of BCAAs and metabolites caused by BCAA supplementation in the subcutaneous white adipose tissue (sWAT) of obese mice. BCAA supplementation reversed insulin sensitivity ameliorated by exercise. The phosphorylation of protein kinase B (Ser473 and Ser474) was decreased by BCAAs in the sWAT of obese mice. However, BCAA supplementation had no such effects in lean mice. BCAAs also increased the expression of fatty acid synthase and other lipogenesis genes in the sWAT of exercised obese mice. BCAA restriction had no effect on body weight and insulin sensitivity in obese mice. CONCLUSIONS: BCAA supplementation impaired the beneficial effect of exercise on glycolipid metabolism in obese but not lean mice. Caution should be taken regarding the use of BCAAs for individuals with obesity who exercise.

Endothelial PPARδ facilitates the post-ischemic vascular repair through interaction with HIF1α.

Rationale: Restoration of vascular perfusion in peripheral arterial disease involves a combination of neovessel formation and the functional restoration of vascular endothelium. Previous studies indicated that ligand-dependent PPARδ activation enhances angiogenesis. However, how PPARδ is triggered by hypoxia and its downstream effects during post-ischemic vascular repair was not well understood. Methods: We induced experimental hindlimb ischemia in endothelial cell selective Ppard knockout induced by Cdh5-Cre mediated deletion of floxed Ppard allele in mice and their wild type control and observed blood perfusion, capillary density, vascular relaxation, and vascular leakage. Results: Deletion of endothelial Ppard delayed perfusion recovery and tissue repair, accompanied by delayed post-ischemic angiogenesis, impaired restoration of vascular integrity, more vascular leakage and enhanced inflammatory responses. At the molecular level, hypoxia upregulated and activated PPARδ in endothelial cells, whereas PPARδ reciprocally stabilized HIF1α protein to prevent its ubiquitin-mediated degradation. PPARδ directly bound to the oxygen-dependent degradation domain of HIF1α at the ligand-dependent domain of PPARδ. Importantly, this HIF1α-PPARδ interaction was independent of PPARδ ligand. Adeno-associated virus mediated endothelium-targeted overexpression of stable HIF1α in vivo improved perfusion recovery, suppressed vascular inflammation, and enhanced vascular repair, to counteract with the effect of Ppard knockout after hindlimb ischemia in mice. Conclusions: In summary, hypoxia-induced, ligand-independent activation of PPARδ in ECs stabilizes HIF1α and serves as a critical regulator for HIF1α activation to facilitate the post-ischemic restoration of vascular homeostasis.

Restoration of Autophagic Flux Improves Endothelial Function in Diabetes Through Lowering Mitochondrial ROS-Mediated eNOS Monomerization.

Endothelial nitric oxide synthase (eNOS) monomerization and uncoupling play crucial roles in mediating vascular dysfunction in diabetes, although the underlying mechanisms are still incompletely understood. Increasing evidence indicates that autophagic dysregulation is involved in the pathogenesis of diabetic endothelial dysfunction; however, whether autophagy regulates eNOS activity through controlling eNOS monomerization or dimerization remains elusive. In this study, autophagic flux was impaired in the endothelium of diabetic db/db mice and in human endothelial cells exposed to advanced glycation end products or oxidized low-density lipoprotein. Inhibition of autophagic flux by chloroquine or bafilomycin A1 were sufficient to induce eNOS monomerization and lower nitric oxide bioavailability by increasing mitochondrial reactive oxygen species (mtROS). Restoration of autophagic flux by overexpressing transcription factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis, decreased endothelial cell oxidative stress, increased eNOS dimerization, and improved endothelium-dependent relaxations (EDRs) in db/db mouse aortas. Inhibition of mammalian target of rapamycin kinase (mTOR) increased TFEB nuclear localization, reduced mtROS accumulation, facilitated eNOS dimerization, and enhanced EDR in db/db mice. Moreover, calorie restriction also increased TFEB expression, improved autophagic flux, and restored EDR in the aortas of db/db mice. Taken together, the findings of this study reveal that mtROS-induced eNOS monomerization is closely associated with the impaired TFEB-autophagic flux axis leading to endothelial dysfunction in diabetic mice.

Endothelial dysfunction after androgen deprivation therapy and the possible underlying mechanisms.

INTRODUCTION: Androgen deprivation therapy (ADT) is a key treatment modality in the management of prostate cancer (PCa), especially for patients with metastatic disease. Increasing evidences suggest that patients who received ADT have increased incidence of diabetes, myocardial infarction, stroke, and even mortality. It is important to understand the pathophysiological mechanisms on how ADT increases cardiovascular risk and induces cardiovascular events, which would provide important information for potential implementation of preventive measures. METHODS: Twenty-six 12-week-old male SD rats were divided into four groups for different types of ADTs including: the bilateral orchidectomy group (Orx), LHRH agonist group (leuprolide), LHRH antagonist group (degarelix), and control group. After treated with drug or adjuvant injection every 3 weeks for 24 weeks, all rats were sacrificed and total blood were collected. Aorta, renal arteries, and kidney were preserved for functional assay, immunohistochemistry, western blot, and quantitative reverse-transcription polymerase chain reaction. RESULTS: In vascular reactivity assays, aorta, intrarenal, and coronary arteries of all three ADT groups showed endothelial dysfunction. AT1R and related molecules at protein and messenger RNA (mRNA) level were tested, and AT1R pathway was shown to be activated and played a role in endothelial dysfunction. Both ACE and AT1R mRNA levels were doubled in the aorta in the leuprolide group while Orx and degarelix groups showed upregulation of AT1R in the kidney tissues. By immunohistochemistry, our result showed higher expression of AT1R in the intrarenal arteries of leuprolide and degarelix groups. The role of reactive oxygen species in endothelial dysfunction was confirmed by DHE fluorescence, nitrotyrosine overexpression, and upregulation of NOX2 in the different ADT treatment groups. CONCLUSION: ADT causes endothelial dysfunction in male rats. GnRH receptor agonist compared to GnRH receptor antagonist, showed more impairment of endothelial function in the aorta and intrarenal arteries. Such change might be associated with upregulation and activation of AngII-AT1R-NOX2 induced oxidative stress in the vasculature. These results help to explain the different cardiovascular risks and outcomes related to different modalities of ADT treatment.

Berberine Reverses Nitroglycerin Tolerance through Suppressing Protein Kinase C Alpha Activity in Vascular Smooth Muscle Cells.

PURPOSE: The aim of this study was to evaluate the effects of berberine on nitroglycerin (NTG) tolerance and explore the underlying mechanism involved. METHODS: NTG tolerance was induced by pre-exposure of Sprague-Dawley rat aortas to NTG in vitro or by pretreating Sprague-Dawley rats with an NTG patch in vivo. The aortas were pre-treated with berberine or PKC inhibitors for different durations of time before induction of NTG tolerance. NTG-induced vasorelaxations was measured on wire myograph. Primary vascular smooth cells (VSMCs) were used to dissect the underlying mechanism of berberine-induced inhibition of NTG tolerance. RESULTS: NTG tolerance induced by either prior exposure of rat aortas to NTG in vitro or pretreatment with an NTG patch in vivo was reversed by co-treatment with berberine, as well as the inhibitors of protein kinase C (PKC) and protein kinase C alpha (PKCα). The mechanistic study revealed that PKCα participated in the development of NTG tolerance as NTG increased the activity of PKCα with enriched PKCα membrane localization and elevated phosphorylation of PKCα in VSMCs, which was reversed by berberine or PKCα inhibitors. CONCLUSION: This study is probably the first demonstration that berberine reverses NTG tolerance through inhibiting PKCα activity in VSMCs and PKCα is an important contributor to the development of NTG tolerance. These new findings suggest that berberine could become a promising drug for prevention of NTG tolerance and that targeting PKCα in VSMCs is likely to be a potential therapeutic strategy for reversal of NTG tolerance in blood vessels.

Outcomes of Partial or Total Cystectomy in Advanced Colon Cancer with Suspected Bladder Invasion-Our 8-Year Experience and Literature Review.

PURPOSE: Advanced colon cancers with bladder invasion pose a heavy burden and challenge towards patients and surgeons. Herein, we report our series with regards to operative and oncological outcomes in our 8 years of experience. METHODS: All patients with advanced colonic tumours and suspected bladder invasion being operated from 2012 to 2020 were included. The histological findings, clinical and oncological outcomes were evaluated. RESULTS: Twenty-two patients were included. Partial cystectomy was performed in 17 of them (77%). No neoadjuvant treatment was prescribed. All preoperative computed tomography (CT) scan showed bladder invasion or colovesical fistula. True tumour invasion to bladder (T4b disease) was confirmed in 17 patients (77%) by histopathology. The 3-year overall survival and recurrence rates were 82% and 9%, respectively. CONCLUSION: En bloc resection of colonic tumour with adherent bladder in advanced colon cancers can achieve a good operative and oncological outcome without neoadjuvant therapy. The relatively low concordance rate between preoperative CT scan and final histopathology may limit the benefit of routine administration of neoadjuvant therapy as it may overtreat and delay subsequent oncological treatment of our patients with possible added morbidity.

Loss of myeloid Bmal1 exacerbates hypertensive vascular remodelling through interaction with STAT6 in mice.

AIMS: In addition to its involvement of inflammatory responses, limited information is available on the phenotype and behaviour of vascular macrophages during hypertensive vascular remodelling. Here, we aim at studying the contribution of BMAL1 to the pro-fibrotic macrophage phenotype in the vasculature during hypertension, which leads to enhanced vascular remodelling and promoted blood pressure increase. METHODS AND RESULTS: Wild type Bmal1f/f and myeloid cell selective Bmal1 knockout Bmal1f/f; LysMCre/+ mice were infused with AngII for 4 weeks to induce hypertension. AngII-induced blood pressure increase, vascular media thickness and vascular dysfunction were enhanced in Bmal1f/f; LysMCre/+ mice, accompanied with a pro-fibrotic M2 phenotype of the vascular macrophages. Bmal1f/f; LysMCre/+ mice also have more up-regulations of MMP9 and MMP13 expression in the vascular wall, accompanied by enhanced collagen deposition after AngII infusion. Loss of Bmal1 in bone marrow-derived macrophages enhanced STAT6 activation induced by IL4, and the subsequent MMP13 up-regulation and activity. In macrophages, loss of Bmal1 enhanced the phosphorylation and nuclear translocation of STAT6 triggered by IL4, through possibly a direct interaction between BMAL1 and STAT6. To further determine whether IL4-induced signalling in macrophage contributes to enhanced vascular remodelling in hypertensive mice, we showed that deletion of myeloid IL4Rα in Il4raf/f; LysMCre/+ mice attenuated blood pressure increase and hypertensive vascular remodelling after AngII infusion. CONCLUSIONS: Our results suggested a tonic effect of BMAL1 deletion on hypertensive vascular remodelling. BMAL1 might inhibit IL4-STAT6 signalling in macrophages through the interaction with STAT6 to reduce STAT6 activation and target gene transcription, especially MMP9 and MMP13, contributing to vascular remodelling.

Salvianolic acid B ameliorates vascular endothelial dysfunction through influencing a bone morphogenetic protein 4-ROS cycle in diabetic mice.

AIM: This study investigated the roles of bone morphogenetic protein-4 (BMP4) and ROS in diabetic endothelial dysfunction and explored whether Salvianolic acid B (Sal B) improved endothelial function by affecting BMP4-ROS in diabetic mice. MAIN METHODS: db/db mice were orally administrated with Sal B (10 mg/kg/day) for one week while db/m + mice were injected with adenoviral vectors delivering BMP4 (3 × 108 pfu) and then received one week-Sal B treatment. ROS levels were assayed by DHE staining. Protein expression and phosphorylation were evaluated by Western blot. Aortic rings were suspended in myograph for force measurement. Flow-mediated dilatations in the second-order mesenteric arteries were determined by pressure myograph. KEY FINDINGS: We first revealed the existence of a BMP4-ROS cycle in db/db mice, which stimulated p38 MAPK/JNK/caspase 3 and thus participated in endothelial dysfunction. One week-treatment or 24 h-incubation with Sal B disrupted the cycle, suppressed p38 MAPK/JNK/caspase 3 cascade, and improved endothelium-dependent relaxations (EDRs) in db/db mouse aortas. Importantly, in vivo Sal B treatment also improved flow-mediated dilatation in db/db mouse second order mesenteric arteries. Furthermore, in vivo BMP4 overexpression induced oxidative stress, stimulated p38 MAPK/JNK/caspase 3, and impaired EDRs in db/m + mouse aortas, which were all reversed by Sal B. SIGNIFICANCE: The present study demonstrates that Sal B ameliorates endothelial dysfunction through breaking the BMP4-ROS cycle and subsequently inhibiting p38 MAPK/JNK/caspase 3 in diabetic mice and provides evidence for the additional new mechanism underlying the benefit of Sal B against diabetic vasculopathy.

KLF2 Mediates the Suppressive Effect of Laminar Flow on Vascular Calcification by Inhibiting Endothelial BMP/SMAD1/5 Signaling.

[Figure: see text].