Urolithin A promotes atherosclerotic plaque stability by limiting inflammation and hypercholesteremia in Apolipoprotein E-deficient mice.

Urolithin A (UroA), a dietary phytochemical, is produced by gut bacteria from fruits rich in natural polyphenols ellagitannins (ETs). The efficiency of ETs metabolism to UroA in humans depends on gut microbiota. UroA has shown a variety of pharmacological activities. In this study we investigated the effects of UroA on atherosclerotic lesion development and stability. Apolipoprotein E-deficient (ApoE-/-) mice were fed a high-fat and high-cholesterol diet for 3 months to establish atherosclerosis model. Meanwhile the mice were administered UroA (50 mg·kg-1·d-1, i.g.). We showed that UroA administration significantly decreased diet-induced atherosclerotic lesions in brachiocephalic arteries, macrophage content in plaques, expression of endothelial adhesion molecules, intraplaque hemorrhage and size of necrotic core, while increased the expression of smooth muscle actin and the thickness of fibrous cap, implying features of plaque stabilization. The underlying mechanisms were elucidated using TNF-α-stimulated human endothelial cells. Pretreatment with UroA (10, 25, 50 µM) dose-dependently inhibited TNF-α-induced endothelial cell activation and monocyte adhesion. However, the anti-inflammatory effects of UroA in TNF-α-stimulated human umbilical vein endothelial cells (HUVECs) were independent of NF-κB p65 pathway. We conducted RNA-sequencing profiling analysis to identify the differential expression of genes (DEGs) associated with vascular function, inflammatory responses, cell adhesion and thrombosis in UroA-pretreated HUVECs. Human disease enrichment analysis revealed that the DEGs were significantly correlated with cardiovascular diseases. We demonstrated that UroA pretreatment mitigated endothelial inflammation by promoting NO production and decreasing YAP/TAZ protein expression and TEAD transcriptional activity in TNF-α-stimulated HUVECs. On the other hand, we found that UroA administration modulated the transcription and cleavage of lipogenic transcription factors SREBP1/2 in the liver to ameliorate cholesterol metabolism in ApoE-/- mice. This study provides an experimental basis for new dietary therapeutic option to prevent atherosclerosis.

Role of Argininosuccinate Synthase 1 -Dependent L-Arginine Biosynthesis in the Protective Effect of Endothelial Sirtuin 3 Against Atherosclerosis.

Atherosclerosis is initiated with endothelial cell (EC) dysfunction and vascular inflammation under hyperlipidemia. Sirtuin 3 (SIRT3) is a mitochondrial deacetylase. However, the specific role of endothelial SIRT3 during atherosclerosis remains poorly understood. The present study aims to study the role and mechanism of SIRT3 in EC function during atherosclerosis. Wild-type Sirt3f/f mice and endothelium-selective SIRT3 knockout Sirt3f/f; Cdh5Cre/+ (Sirt3EC-KO) mice are injected with adeno-associated virus (AAV) to overexpress PCSK9 and fed with high-cholesterol diet (HCD) for 12 weeks to induce atherosclerosis. Sirt3EC-KO mice exhibit increased atherosclerotic plaque formation, along with elevated macrophage infiltration, vascular inflammation, and reduced circulating L-arginine levels. In human ECs, SIRT3 inhibition resulted in heightened vascular inflammation, reduced nitric oxide (NO) production, increased reactive oxygen species (ROS), and diminished L-arginine levels. Silencing of SIRT3 results in hyperacetylation and deactivation of Argininosuccinate Synthase 1 (ASS1), a rate-limiting enzyme involved in L-arginine biosynthesis, and this effect is abolished in mutant ASS1. Furthermore, L-arginine supplementation attenuates enhanced plaque formation and vascular inflammation in Sirt3EC-KO mice. This study provides compelling evidence supporting the protective role of endothelial SIRT3 in atherosclerosis and also suggests a critical role of SIRT3-induced deacetylation of ASS1 by ECs for arginine synthesis.

Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells Enhance the Osteoblastic Differentiation of Periodontal Ligament Stem Cells Under High Glucose Conditions Through the PI3K/AKT Signaling Pathway.

Objective: High glucose (HG) can influence the osteogenic differentiation ability of periodontal ligament stem cells (PDLSCs). Human umbilical cord mesenchymal stem cell-derived exosomes (hUCMSC-exo) have broad application prospects in tissue healing. The current study aimed to explore whether hUCMSC-exo could promote the osteogenic differentiation of hPDLSCs under HG conditions and the underlying mechanism. Methods: We used a 30 mmol/L glucose concentration to simulate HG conditions. CCK-8 assay was performed to evaluate the effect of hUCMSC-exo on the proliferation of hPDLSCs. Alkaline phosphatase (ALP) staining, ALP activity, and qRT-PCR were performed to evaluate the pro-osteogenic effect of hUCMSC-exo on hPDLSCs. Western blot analysis was conducted to evaluate the underlying mechanism. Results: The results of the CCK-8 assay, ALP staining, ALP activity, and qRT-PCR assay showed that hUCMSC-exo significantly promoted cell proliferation and osteogenic differentiation in a dose-dependent manner. The Western blot results revealed that hUCMSC-exo significantly increased the levels of p-PI3K and p-AKT in cells, and the effect was inhibited by LY294002 (PI3K inhibitor) or MK2206 (AKT inhibitor), respectively. Moreover, the increases in osteogenic indicators induced by hUCMSC-exo were significantly suppressed by LY294002 and MK2206. Conclusion: hUCMSC-exo promote the osteogenic differentiation of hPDLSCs under HG conditions through the PI3K/AKT signaling pathway.

Sourcing of human peripheral blood-derived myeloid angiogenic cells under xeno-free conditions for the treatment of critical limb ischemia.

BACKGROUND: Critical limb ischemia (CLI) is the most severe form of peripheral artery disease and exhibits a high risk of lower extremity amputations. As even the most promising experimental approaches based on mesenchymal stem cells (MSCs) demonstrated only moderate therapeutic effects, we hypothesized that other cell types with intrinsic roles in angiogenesis may exhibit a stronger therapeutic potential. We have previously established a protocol to source human peripheral blood-derived angiogenic cells (BDACs). These cells promoted revascularization and took perivascular location at sites of angiogenesis, thus resembling hematopoietic pericytes, which were only described in vivo so far. We thus hypothesized that BDACs might have a superior ability to promote revascularization and rescue the affected limb in CLI. METHODS: As standard BDAC sourcing techniques involve the use of animal-derived serum, we sought to establish a xeno- and/or serum-free protocol. Next, BDACs or MSCs were injected intramuscularly following the ligation of the iliac artery in a murine model. Their ability to enhance revascularization, impair necrosis and modulate inflammatory processes in the affected limb was investigated. Lastly, the secretomes of both cell types were compared to find potential indications for the observed differences in angiogenic potential. RESULTS: From the various commercial media tested, one xeno-free medium enabled the derivation of cells that resembled functional BDACs in comparable numbers. When applied to a murine model of CLI, both cell types enhanced limb reperfusion and reduced necrosis, with BDACs being twice as effective as MSCs. This was also reflected in histological evaluation, where BDAC-treated animals exhibited the least muscle tissue degeneration. The BDAC secretome was enriched in a larger number of proteins with pro-angiogenic and anti-inflammatory properties, suggesting that the combination of those factors may be responsible for the superior therapeutic effect. CONCLUSIONS: Functional BDACs can be sourced under xeno-free conditions paving the way for their safe clinical application. Since BDACs are derived from an easily accessible and renewable tissue, can be sourced in clinically relevant numbers and time frame and exceeded traditional MSCs in their therapeutic potential, they may represent an advantageous cell type for the treatment of CLI and other ischemic diseases.

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.

Endothelium-targeted delivery of PPARδ by adeno-associated virus serotype 1 ameliorates vascular injury induced by hindlimb ischemia in obese mice.

Diabetic vasculopathy is a major health problem worldwide. Peripheral arterial disease (PAD), and in its severe form, critical limb ischemia is a major form of diabetic vasculopathy with limited treatment options. Existing literature suggested an important role of PPARδ in vascular homeostasis. It remains elusive for using PPARδ as a potential therapeutic target due to mostly the side effects of PPARδ agonists. To explore the roles of PPARδ in endothelial homeostasis, endothelial cell (EC) selective Ppard knockout and controlled mice were subjected to hindlimb ischemia (HLI) injury. The muscle ECs were sorted for single-cell RNA sequencing (scRNA-seq) analysis. HLI was also performed in high fat diet (HFD)-induced obese mice to examine the function of PPARδ in obese mice with delayed vascular repair. Adeno-associated virus type 1 (AAV1) carrying ICAM2 promoter to target endothelium for overexpressing PPARδ was injected into the injured muscles of normal chow- and HFD-fed obese mice before HLI surgery was performed. scRNA-seq analysis of ECs in ischemic muscles revealed a pivotal role of PPARδ in endothelial homeostasis. PPARδ expression was diminished both after HLI injury, and also in obese mice, which showed further delayed vascular repair. Endothelium-targeted delivery of PPARδ by AAV1 improved perfusion recovery, increased capillary density, restored endothelial integrity, suppressed vascular inflammation, and promoted muscle regeneration in ischemic hindlimbs of both lean and obese mice. Our study indicated the effectiveness of endothelium-targeted PPARδ overexpression for restoring functional vasculature after ischemic injury, which might be a promising option of gene therapy to treat PAD and CLI.

Intranuclear Delivery of DNA Nanostructures via Cellular Mechanotransduction.

DNA nanostructures are attractive gene carriers for nanomedicine applications, yet their delivery to the nucleus remains inefficient. We present the application of extracellular mechanical stimuli to activate cellular mechanotransduction for boosting the intranuclear delivery of DNA nanostructures. Treating mammalian cells with polythymidine-rich spherical nucleic acids (poly(T) SNAs) under gentle compression by a single coverslip leads to up to ∼50% nuclear accumulation without severe endosomal entrapment, cytotoxicity, or long-term membrane damage; no chemical modification or transfection reagent is needed. Gentle compression activates Rho-ROCK mechanotransduction and causes nuclear translocation of YAP. Joint compression and treatment with poly(T) oligonucleotides upregulate genes linked to myosin, actin filament, and nuclear import. In turn, Rho-ROCK, myosin, and importin mediate the nuclear entry of poly(T) SNAs. Treatment of endothelioma cells with poly(T) SNAs bearing antisense oligonucleotides under compression inhibits an intranuclear oncogene. Our data should inspire the marriage of DNA nanotechnology and cellular biomechanics for intranuclear applications.

Autotaxin signaling facilitates ß cell dedifferentiation and dysfunction induced by Sirtuin 3 deficiency.

OBJECTIVE: ß cell dedifferentiation may underlie the reversible reduction in pancreatic ß cell mass and function in type 2 diabetes (T2D). We previously reported that ß cell-specific Sirt3 knockout (Sirt3f/f;Cre/+) mice developed impaired glucose tolerance and glucose-stimulated insulin secretion after feeding with high fat diet (HFD). RNA sequencing showed that Sirt3-deficient islets had enhanced expression of Enpp2 (Autotaxin, or ATX), a secreted lysophospholipase which produces lysophosphatidic acid (LPA). Here, we hypothesized that activation of the ATX/LPA pathway contributed to pancreatic ß cell dedifferentiation in Sirt3-deficient ß cells. METHODS: We applied LPA, or lysophosphatidylcoline (LPC), the substrate of ATX for producing LPA, to MIN6 cell line and mouse islets with altered Sirt3 expression to investigate the effect of LPA on ß cell dedifferentiation and its underlying mechanisms. To examine the pathological effects of ATX/LPA pathway, we injected the ß cell selective adeno-associated virus (AAV-Atx-shRNA) or negative control AAV-scramble in Sirt3f/f and Sirt3f/f;Cre/+ mice followed by 6-week of HFD feeding. RESULTS: In Sirt3f/f;Cre/+ mouse islets and Sirt3 knockdown MIN6 cells, ATX upregulation led to increased LPC with increased production of LPA. The latter not only induced reversible dedifferentiation in MIN6 cells and mouse islets, but also reduced glucose-stimulated insulin secretion from islets. In MIN6 cells, LPA induced phosphorylation of JNK/p38 MAPK which was accompanied by ß cell dedifferentiation. The latter was suppressed by inhibitors of LPA receptor, JNK, and p38 MAPK. Importantly, inhibiting ATX in vivo improved insulin secretion and reduced ß cell dedifferentiation in HFD-fed Sirt3f/f;Cre/+ mice. CONCLUSIONS: Sirt3 prevents ß cell dedifferentiation by inhibiting ATX expression and upregulation of LPA. These findings support a long-range signaling effect of Sirt3 which modulates the ATX-LPA pathway to reverse ß cell dysfunction associated with glucolipotoxicity.

Cannabidiol Promotes Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in the Inflammatory Microenvironment via the CB2-dependent p38 MAPK Signaling Pathway.

Background and Objectives: Chronic inflammation of bone tissue often results in bone defects and hazards to tissue repair and regeneration. Cannabidiol (CBD) is a natural cannabinoid with multiple biological activities, including anti-inflammatory and osteogenic potential. This study aimed to investigate the efficacy and mechanisms of CBD in the promotion of bone marrow mesenchymal stem cells (BMSCs) osteogenic differentiation in the inflammatory microenvironment. Methods and Results: BMSCs isolated from C57BL/6 mice, expressed stem cell characteristic surface markers and presented multidirectional differentiation potential. The CCK-8 assay was applied to evaluate the effects of CBD on BMSCs' vitality, and demonstrating the safety of CBD on BMSCs. Then, BMSCs were stimulated with lipopolysaccharide (LPS) to induce inflammatory microenvironment. We found that CBD intervention down-regulated mRNA expression levels of inflammatory cytokines and promoted cells proliferation in LPS-treated BMSCs, also reversed the protein and mRNA levels downregulation of osteogenic markers caused by LPS treatment. Moreover, CBD intervention activated the cannabinoid receptor 2 (CB2) and the p38 mitogen-activated protein kinase (MAPK) signaling pathway. While AM630, a selective CB2 inhibitor, reduced phosphorylated (p)-p38 levels. In addition, AM630 and SB530689, a selective p38 MAPK inhibitor, attenuated the enhancement of osteogenic markers expression levels by CBD in inflammatory microenvironment, respectively. Conclusions: CBD promoted osteogenic differentiation of BMSCs via the CB2/p38 MAPK signaling pathway in the inflammatory microenvironment.

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.

Type 2 innate immunity drives distinct neonatal immune profile conducive for heart regeneration.

Aims: Neonatal immunity is functionally immature and skewed towards a TH2-driven, anti-inflammatory profile. This neonatal immunotolerance is partly driven by the type 2 cytokines: interleukin-4 (IL-4) and interleukin-13 (IL-13). Studies on neonatal cardiac regeneration reveal the beneficial role of an anti-inflammatory response in restoring cardiac function after injury. However, the role of an imbalanced immune repertoire observed in neonates on tissue regeneration is poorly understood; specifically, whether IL-4 and IL-13 actively modulate neonatal immunity during cardiac injury. Methods and results: Neonatal mice lacking IL-4 and IL-13 (DKOs) examined at 2 days after birth exhibited reduced anti-inflammatory immune populations with basal cardiac immune populations like adult mice. Examination of neonates lacking IL-4 and IL-13 at 2 days post cardiac ischemic injury, induced on the second day after birth, showed impaired cardiac function compared to their control counterparts. Treatment with either IL-4 or IL-13 cytokine during injury restored both cardiac function and immune population profiles in knockout mice. Examination of IL-4/IL-13 downstream pathways revealed the role of STAT6 in mediating the regenerative response in neonatal hearts. As IL-4/IL-13 drives polarization of alternatively activated macrophages, we also examined the role of IL-4/IL-13 signaling within the myeloid compartment during neonatal cardiac regeneration. Injury of IL-4Rα myeloid specific knockout neonates 2 days after birth revealed that loss of IL-4/IL-13 signaling in macrophages alone was sufficient to impair cardiac regeneration. Conclusions: Our results confirm that the TH2 cytokines: IL-4 and IL-13, which skews neonatal immunity to a TH2 profile, are necessary for maintaining and mediating an anti-inflammatory response in the neonatal heart, in part through the activation of alternatively activated macrophages, thereby permitting a niche conducive for regeneration.

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.

REV-ERBα agonist SR9009 suppresses IL-1ß production in macrophages through BMAL1-dependent inhibition of inflammasome.

The circadian clock plays an important role in adapting organisms to the daily light/dark cycling environment. Recent research findings reveal the involvement of the circadian clock not only in physiological functions but also in regulating inflammatory responses under pathological situations. Previous studies showed that the time-of-day variance of leucocyte circulation and pro-inflammatory cytokines secretion could be directly regulated by the clock-related proteins, including BMAL1 and REV-ERBα in a 24-hour oscillation pattern. To investigate the molecular mechanism behind the regulation of inflammation by the core clock components, we focus on the inflammatory responses in macrophages. Using bone marrow-derived macrophages from wild type and myeloid selective BMAL1-knockout mice, we found that the production of inflammatory cytokines, particularly IL-1ß, was dependent on the timing of the lipopolysaccharide (LPS) stimulation in macrophages. Pharmacological activation of REV-ERBα with SR9009 significantly suppressed the LPS-induced inflammation in vitro and in vivo. Particularly, the effect of SR9009 on inhibiting NLRP3-mediated IL-1ß and IL-18 production in macrophages was dependent on BMAL1 expression. Further analysis of the metabolic activity in LPS-treated mice showed that knockout of BMAL1 in macrophages exacerbated the hypometabolic state and delayed the recovery from LPS-induced endotoxemia even in the presence of SR9009. These results demonstrated an anti-inflammatory role of REV-ERBα in endotoxin-induced inflammation, during which the secretion of IL-1ß through the NLRP3 inflammasome pathway inhibited by SR9009 was regulated by BMAL1.

Punicalagin Attenuates Disturbed Flow-Induced Vascular Dysfunction by Inhibiting Force-Specific Activation of Smad1/5.

BACKGROUND: Pathophysiological vascular remodeling in response to disturbed flow with low and oscillatory shear stress (OSS) plays important roles in atherosclerosis progression. Pomegranate extraction (PE) was reported having anti-atherogenic effects. However, whether it can exert a beneficial effect against disturbed flow-induced pathophysiological vascular remodeling to inhibit atherosclerosis remains unclear. The present study aims at investigating the anti-atherogenic effects of pomegranate peel polyphenols (PPP) extraction and its purified compound punicalagin (PU), as well as their protective effects on disturbed flow-induced vascular dysfunction and their underlying molecular mechanisms. METHODS: The anti-atherogenic effects of PPP/PU were examined on low-density lipoprotein receptor knockout mice fed with a high fat diet. The vaso-protective effects of PPP/PU were examined in rat aortas using myograph assay. A combination of in vivo experiments on rats and in vitro flow system with human endothelial cells (ECs) was used to investigate the pharmacological actions of PPP/PU on EC dysfunction induced by disturbed flow. In addition, the effects of PPP/PU on vascular smooth muscle cell (VSMC) dysfunction were also examined. RESULTS: PU is the effective component in PPP against atherosclerosis. PPP/PU evoked endothelium-dependent relaxation in rat aortas. PPP/PU inhibited the activation of Smad1/5 in the EC layers at post-stenotic regions of rat aortas exposed to disturbed flow with OSS. PPP/PU suppressed OSS-induced expression of cell cycle regulatory and pro-inflammatory genes in ECs. Moreover, PPP/PU inhibited inflammation-induced VSMC dysfunction. CONCLUSION: PPP/PU protect against OSS-induced vascular remodeling through inhibiting force-specific activation of Smad1/5 in ECs and this mechanism contributes to their anti-atherogenic effects.

Increased co-expression of PSMA2 and GLP-1 receptor in cervical cancer models in type 2 diabetes attenuated by Exendin-4: A translational case-control study.

BACKGROUND: Type 2 diabetes (T2D) increases the risk of many types of cancer. Dysregulation of proteasome-related protein degradation leads to tumorigenesis, while Exendin-4, a glucagon-like peptide 1 receptor (GLP-1R) agonist, possesses anti-cancer effects. METHODS: We explored the co-expression of proteasome alpha 2 subunit (PSMA2) and GLP-1R in the Cancer Genome Atlas (TCGA) database and human cervical cancer specimens, supplemented by in vivo and in vitro studies using multiple cervical cancer cell lines. FINDINGS: PSMA2 expression was increased in 12 cancer types in TCGA database and cervical cancer specimens from patients with T2D (T2D vs non-T2D: 3.22 (95% confidence interval CI: 1.38, 5.05) vs 1.00 (0.66, 1.34) fold change, P = 0.01). psma2-shRNA decreased cell proliferation in vitro, and tumour volume and Ki67 expression in vivo. Exendin-4 decreased psma2 expression, tumour volume and Ki67 expression in vivo. There was no change in GLP-1R expression in 12 cancer types in TCGA database. However, GLP-1R expression (T2D vs non-T2D: 5.49 (3.0, 8.1) vs 1.00 (0.5, 1.5) fold change, P < 0.001) was increased and positively correlated with PSMA2 expression in T2D-related (r = 0.68)  but not in non-T2D-related cervical cancer specimens. This correlation was corroborated by in vitro experiments where silencing glp-1r decreased psma2 expression. Exendin-4 attenuated phospho-p65 and -IκB expression in the NF-κB pathway. INTERPRETATION: PSMA2 and GLP-1R expression in T2D-related cervical cancer specimens was increased and positively correlated, suggesting hyperglycaemia might promote cancer growth by increasing PSMA2 expression which could be attenuated by Exendin-4. FUNDING: This project was supported by Postdoctoral Fellowship Scheme, Direct Grant, Diabetes Research and Education Fund from the Chinese University of Hong Kong (CUHK).

A GLP-1 analog lowers ER stress and enhances protein folding to ameliorate homocysteine-induced endothelial dysfunction.

Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular diseases and increases mortality in type 2 diabetic patients. HHcy induces endoplasmic reticulum (ER) stress and oxidative stress to impair endothelial function. The glucagon-like peptide 1 (GLP-1) analog exendin-4 attenuates endothelial ER stress, but the detailed vasoprotective mechanism remains elusive. The present study investigated the beneficial effects of exendin-4 against HHcy-induced endothelial dysfunction. Exendin-4 pretreatment reversed homocysteine-induced impairment of endothelium-dependent relaxations in C57BL/6 mouse aortae ex vivo. Four weeks subcutaneous injection of exendin-4 restored the impaired endothelial function in both aortae and mesenteric arteries isolated from mice with diet-induced HHcy. Exendin-4 treatment lowered superoxide anion accumulation in the mouse aortae both ex vivo and in vivo. Exendin-4 decreased the expression of ER stress markers (e.g., ATF4, spliced XBP1, and phosphorylated eIF2α) in human umbilical vein endothelial cells (HUVECs), and this change was reversed by cotreatment with compound C (CC) (AMPK inhibitor). Exendin-4 induced phosphorylation of AMPK and endothelial nitric oxide synthase in HUVECs and arteries. Exendin-4 increased the expression of endoplasmic reticulum oxidoreductase (ERO1α), an important ER chaperone in endothelial cells, and this effect was mediated by AMPK activation. Experiments using siRNA-mediated knockdown or adenoviral overexpression revealed that ERO1α mediated the inhibitory effects of exendin-4 on ER stress and superoxide anion production, thus ameliorating HHcy-induced endothelial dysfunction. The present results demonstrate that exendin-4 reduces HHcy-induced ER stress and improves endothelial function through AMPK-dependent ERO1α upregulation in endothelial cells and arteries. AMPK activation promotes the protein folding machinery in endothelial cells to suppress ER stress.

TSG-6 Inhibits the Growth of Keloid Fibroblasts Via Mediating the TGF-ß1/Smad Signaling Pathway.

BACKGROUND: The cytokine TNF-α-stimulated gene-6 (TSG-6) had been verified to have a certain inhibitory effect on the inflammation. During wound healing, fibroblasts increasingly proliferated and deposited collagen fibers, leading to the formation of pathological scars. We sought to elucidate the mechanism by which the TGF-ß1/Smad pathway was mediated by TSG-6 in human keloid fibroblasts. MATERIALS AND METHODS: Human keloid fibroblast cells were isolated from keloid tissue by enzyme digestion and identified by immunocytochemistry. Lentiviral vectors pLVX-puro-TSG-6 and pLVX-shRNA1-TSG-6 were constructed which were then transfected into human keloid fibroblasts. The mRNA and protein levels of TSG-6 were detected respectively by RT-PCR and western blot assay. The intracellular localization of TGF-ß1-induced proteins and phosphorylated (p)-Smad2/3 in keloid fibroblasts were investigated using an immunofluorescence assay. Plasminogen activator inhibitor-1 (PAI-1) transcriptional activity was detected by RT-PCR. RESULTS: TSG-6 could effectively interfere the TGF-ß1/Smad signal transduction pathway in keloid fibroblasts rather than in normal fibroblasts. The phosphorylation levels of Smad2/3 were notably reduced by TSG-6 treatment. TSG-6 blocked the complex formation of Smad2/3/4, and their nuclear translocation. However, it upregulated Smad7 expression, presenting dose dependence. PAI-1 was also suppressed by TSG-6 treatment. CONCLUSIONS: TSG-6 inhibits proliferation by inducing apoptosis in keloid fibroblasts, which may be associated with TGF-ß1/Smad pathway.

Endothelial Agrin Is Dispensable for Normal and Tumor Angiogenesis.

Recently, the extracellular matrix protein agrin has been reported to promote tumor angiogenesis that supports tumorigenesis and metastasis; however, there is a lack of in vivo genetic evidence to prove whether agrin derived from the tumors or endothelial cells (ECs) systemically should be the therapeutic target. To date, the physiological role of endothelial agrin has also not been investigated. In the EC-specific agrin knockout mice, we observed normal endothelial and haematopoietic cell development during embryogenesis. Moreover, these mice develop normal vascular barrier integrity and vasoreactivity at the adult stage. Importantly, the growth of localized or metastatic cancer cells was not affected after implantation into endothelial agrin depleted mice. Mechanistically, agrin did not regulate endothelial ERK1/2, YAP or p53 activation in vivo that is central to support endothelial proliferation, survival and invasion. Cumulatively, our findings may suggest that agrin could play a redundant role in endothelial development during physiological and tumor angiogenesis. Targeting the endothelial derived agrin might not be effective in inhibiting tumor angiogenesis.

Associations of Sedentary Patterns with Cardiometabolic Biomarkers in Physically Active Young Males.

PURPOSE: Sitting time (ST) is a serious global health issue and positively associated with cardiometabolic disease. The present study investigated associations between objectively measured ST, sedentary patterns, and cardiometabolic biomarkers in physically active young males. METHODS: Cross-sectional analysis was completed in 94 males 18-35 yr of age. Total ST, prolonged sedentary bouts (≥30 min with no interruption), and sedentary breaks (transitions from sitting/lying to standing/stepping) were assessed using activPAL. Lipids, insulin, C-peptide, C-reactive protein (CRP), vascular cellular adhesion molecule-1, intercellular adhesion molecule 1, E-selectin, P-selectin, leptin, resistin, and adiponectin were measured using assay kits. The expression of specific proteins related to endothelial dysfunction was determined using quantitative real-time polymerase chain reaction. Associations between total ST, prolonged sedentary bouts, and sedentary breaks with cardiometabolic biomarkers and total ST and levels of gene expression were assessed using generalized linear models. RESULTS: Total ST was significantly associated with triglycerides (B = 1.814), insulin (B = 2.117), homeostasis model assessment of insulin resistance (B = 0.071), and E-selectin (B = 2.052). Leptin (B = 0.086), E-selectin (B = 1.623), and P-selectin (B = 2.519) were significantly associated with prolonged sedentary bouts, whereas leptin (B = -0.017) and CRP (B = -0.016) were associated with sedentary breaks. After adjustment for moderate to vigorous physical activity, the associations between triglycerides (B = 2.048) and total ST, and between CRP (B = -0.016) and sedentary breaks, remained significant. E-selectin mRNA levels (B = 0.0002) were positively associated with ST with or without adjustment for moderate to vigorous physical activity. CONCLUSIONS: Total ST and prolonged sedentary bouts were positively associated with several cardiometabolic biomarkers, with interruptions in ST potentially contributing to reduced cardiometabolic risk in physically active young male adults.

The Role of Macrophages in Vascular Repair and Regeneration after Ischemic Injury.

Macrophage is one of the important players in immune response which perform many different functions during tissue injury, repair, and regeneration. Studies using animal models of cardiovascular diseases have provided a clear picture describing the effect of macrophages and their phenotype during injury and regeneration of various vascular beds. Many data have been generated to demonstrate that macrophages secrete many important factors including cytokines and growth factors to regulate angiogenesis and arteriogenesis, acting directly or indirectly on the vascular cells. Different subsets of macrophages may participate at different stages of vascular repair. Recent findings also suggest a direct interaction between macrophages and other cell types during the generation and repair of vasculature. In this short review, we focused our discussion on how macrophages adapt to the surrounding microenvironment and their potential interaction with other cells, in the context of vascular repair supported by evidences mostly from studies using hindlimb ischemia as a model for studying post-ischemic vascular repair.