Fabrication of plumbagin on silver nanoframework for tunable redox modulation: Implications for therapeutic angiogenesis

The redox state of the endothelial cells plays a key role in the regulation of the angiogenic process. The modulation of the redox state of endothelial cells (ECs) could be a viable target to alter angiogenic response. In the present work, we synthesized a redox modulator by caging 5-hydroxy 2-methyl 1, 4-napthoquinone (Plumbagin) on silver nano framework (PCSN) for tunable reactive oxygen species (ROS) inductive property and tested its role in ECs during angiogenic response in physiological and stimulated conditions. In physiological conditions, the redox modulators induced the angiogenic response by establishing ECs cell–cell contact in tube formation model, chorio allontoic membrane, and aortic ring model. The molecular mechanism of angiogenic response was induced by vascular endothelial growth factor receptor 2 (VEGFR2)/p42-mitogen-activated protein kinase signaling pathway. Under stimulation, by mimicking tumor angiogenic conditions it induced cytotoxicity by generation of excessive ROS and inhibited the angiogenic response by the loss of spatiotemporal regulation of matrix metalloproteases, which prevents the tubular network formation in ECs and poly-ADP ribose modification of VEGF. The mechanism of opposing effects of PCSN was due to modulation of PKM2 enzyme activity, which increased the EC sensitivity to ROS and inhibited EC survival in stimulated condition. In normal conditions, the endogenous reactive states of NOX4 enzyme helped the EC survival. The results indicated that a threshold ROS level exists in ECs that promote angiogenesis and any significant enhancement in its level by redox modulator inhibits angiogenesis. The study provides the cues for the development of redox-based therapeutic molecules to cure the disease-associated aberrant angiogenesis.     

Role of VEGFR‐1 in melanoma acquired resistance to the BRAF inhibitor vemurafenib

The vascular endothelial growth factor receptor‐1 (VEGFR‐1) is a tyrosine kinase receptor frequently expressed in melanoma. Its activation by VEGF‐A or placental growth factor (PlGF) promotes tumour cell survival, migration and invasiveness. Moreover, VEGFR‐1 stimulation contributes to pathological angiogenesis and induces recruitment of tumour‐associated macrophages. Since melanoma acquired resistance to BRAF inhibitors (BRAFi) has been associated with activation of pro‐angiogenic pathways, we have investigated VEGFR‐1 involvement in vemurafenib resistance. Results indicate that human melanoma cells rendered resistant to vemurafenib secrete greater amounts of VEGF‐A and express higher VEGFR‐1 levels compared with their BRAFi‐sensitive counterparts. Transient VEGFR‐1 silencing in susceptible melanoma cells delays resistance development, whereas in resistant cells it increases sensitivity to the BRAFi. Consistently, enforced VEGFR‐1 expression, by stable gene transfection in receptor‐negative melanoma cells, markedly reduces sensitivity to vemurafenib. Moreover, melanoma cells expressing VEGFR‐1 are more invasive than VEGFR‐1 deficient cells and receptor blockade by a specific monoclonal antibody (D16F7 mAb) reduces extracellular matrix invasion triggered by VEGF‐A and PlGF. These data suggest that VEGFR‐1 up‐regulation might contribute to melanoma progression and spreading after acquisition of a drug‐resistant phenotype. Thus, VEGFR‐1 inhibition with D16F7 mAb might be a suitable adjunct therapy for VEGFR‐1 positive tumours with acquired resistance to vemurafenib.     

Vascular endothelial growth factor‐D modulates oxidant–antioxidant balance of human vascular endothelial cells

Vascular endothelial growth factor‐D (VEGF‐D) is an angiogenic and lymphangiogenic glycoprotein that facilitates tumour growth and distant organ metastasis. Our previous studies showed that VEGF‐D stimulates the expression of proteins involved in cell–matrix interactions and promoting the migration of endothelial cells. In this study, we focused on the redox homoeostasis of endothelial cells, which is significantly altered in the process of tumour angiogenesis. Our analysis revealed up‐regulated expression of proteins that form the antioxidant barrier of the cell in VEGF‐D‐treated human umbilical endothelial cells and increased production of reactive oxygen and nitrogen species in addition to a transient elevation in the total thiol group content. Despite a lack of changes in the total antioxidant capacity, modification of the antioxidant barrier induced by VEGF‐D was sufficient to protect cells against the oxidative stress caused by hypochlorite and paraquat. These results suggest that exogenous stimulation of endothelial cells with VEGF‐D induces an antioxidant response of cells that maintains the redox balance. Additionally, VEGF‐D‐induced changes in serine/threonine kinase mTOR shuttling between the cytosol and nucleus and its increased phosphorylation at Ser‐2448, lead us to the conclusion that the observed shift in redox balance is regulated via mTOR kinase signalling.     

Molecular MRI assessment of vascular endothelial growth factor receptor‐2 in rat C6 gliomas

Angiogenesis is essential to tumour progression and a precise evaluation of angiogenesis is important for tumour early diagnosis and treatment. The quantitative and dynamic in vivo assessment of tumour angiogenesis can be achieved by molecular magnetic resonance imaging (mMRI). Vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) are the main regulatory systems in angiogenesis and have been used as hot targets for radionuclide‐based molecular imaging. However, little research has been accomplished in targeting VEGF/VEGFRs by mMRI. In our study, we aimed to assess the expression of VEGFR2 in C6 gliomas by using a specific molecular probe with mMRI. The differential uptake of the probe conjugated to anti‐VEGFR2 monoclonal antibody, shown by varied increases in T₁ signal intensity during a 2 hr period, demonstrated the heterogeneous expression of VEGFR2 in different tumour regions. Microscopic fluorescence imaging, obtained for the biotin group in the probe with streptavidin‐Cy3, along with staining for cellular VEGFR2 levels, laminin and CD45, confirmed the differential distribution of the probe which targeted VEGFR2 on endothelial cells. The angiogenesis process was also assessed using magnetic resonance angiography, which quantified tumour blood volume and provided a macroscopic view and a dynamic change of the correlation between tumour vasculature and VEGFR2 expression. Together these results suggest mMRI can be very useful in assessing and characterizing the expression of specific angiogenic markers in vivo and help evaluate angiogenesis associated with tumour progression.     

HMGB1‐induced angiogenesis in perforated disc cells of human temporomandibular joint

High mobility group 1 protein (HMGB1), a highly conserved nuclear DNA‐binding protein and inflammatory mediator, has been recently found to be involved in angiogenesis. Our previous study has demonstrated the elevation of HMGB1 in the tissue of perforated disc of temporomandibular joint (TMJ). Here, we investigated a novel mediator of HMGB1 in regulating hypoxia‐inducible factor‐1α (HIF‐1α) and vascular endothelial growth factor (VEGF) to mediate angiogenesis in perforated disc cells of TMJ. HMGB1 increased the expression of HIF‐1α and VEGF in a dose‐ and time‐dependent manner in these cells. Moreover, immunofluorescence assay exhibits that the HIF‐1α were activated by HMGB1. In addition, HMGB1 activated extracellular signal‐related kinase 1/2 (Erk1/2), Jun N‐terminal kinase (JNK), but not P38 in these cells. Furthermore, both U0126 (ErK inhibitor) and SP600125 (JNK inhibitor) significantly suppressed the enhanced production of HIF‐1α and VEGF induced by HMGB1. Tube formation of human umbilical vein endothelial cells (HUVECs) was significantly increased by exposure to conditioned medium derived from HMGB1‐stimulated perforated disc cells, while attenuated with pre‐treatment of inhibitors for VEGF, HIF‐1α, Erk and JNK, individually. Therefore, abundance of HMGB1 mediates activation of HIF‐1α in disc cells via Erk and JNK pathway and then, initiates VEGF secretion, thereby leading to disc angiogenesis and accelerating degenerative change of the perforated disc.     

S100A9 gene silencing inhibits the release of pro‐inflammatory cytokines by blocking the IL‐17 signalling pathway in mice with acute pancreatitis

The study aimed to investigate whether S100A9 gene silencing mediating the IL‐17 pathway affected the release of pro‐inflammatory cytokines in acute pancreatitis (AP). Kunming mice were assigned to the normal, AP, AP + negative control (NC), AP + shRNA, AP + IgG and AP + anti IL‐17 groups. ELISA was applied to measure expressions of AMY, LDH, CRP, TNF‐α, IL‐6 and IL‐8. The cells were distributed into the control, blank, NC, shRNA1 and shRNA2 groups. MTT assay, flow cytometry, RT‐qPCR and Western blotting were used to evaluate cell proliferation, cell cycle and apoptosis, and expressions of S100A9, TLR4, RAGE, IL‐17, HMGB1 and S100A12 in tissues and cells. Compared with the normal group, the AP group displayed increased expressions of AMY, LDH, CRP, TNFα, IL‐6, IL‐8, S100A9, TLR4, RAGE, IL‐17, HMGB1 and S100A12. The AP + shRNA and AP + anti IL‐17 groups exhibited an opposite trend. The in vivo results: Compare with the control group, the blank, NC, shRNA1 and shRNA2 groups demonstrated increased expressions of S100A9, TLR4, RAGE, IL‐17, HMGB1 and S100A12, as well as cell apoptosis and cells at the G1 phase, with reduced proliferation. Compared with the blank and NC groups, the shRNA1 and shRNA2 groups had declined expressions of S100A9, TLR4, RAGE, IL‐17, HMGB1 and S100A12, as well as cell apoptosis and cells at the G1 phase, with elevated proliferation. The results indicated that S100A9 gene silencing suppressed the release of pro‐inflammatory cytokines through blocking of the IL‐17 pathway in AP.     

TLR4 Deters Perfusion Recovery and Upregulates Toll-like Receptor 2 (TLR2) in Ischemic Skeletal Muscle and Endothelial Cells

Toll-like receptors (TLRs) play an important role in regulating muscle regeneration and angiogenesis in response to ischemia. TLR2 knockout mice exhibit pronounced skeletal muscle necrosis and abnormal vessel architecture after femoral artery ligation, suggesting that TLR2 signaling is protective during ischemia. TLR4, an important receptor in inflammatory signaling, has been shown to regulate TLR2 expression in other systems. We hypothesize that a similar relationship between TLR4 and TLR2 may exist in hindlimb ischemia in which TLR4 upregulates TLR2, a mediator of angiogenesis and perfusion recovery. We examined the expression of TLR2 in unstimulated and in TLR-agonist treated endothelial cells (ECs). TLR2 expression (low in control ECs) was upregulated by lipopolysaccharide, the danger signal high mobility group box-1, and hypoxia in a TLR4-dependent manner. Endothelial tube formation on Matrigel as well as EC permeability was assessed as in vitro measures of angiogenesis. Time-lapse imaging demonstrated that ECs lacking TLR4 formed more tubes, whereas TLR2 knockdown ECs exhibited attenuated tube formation. TLR2 also mediated EC permeability, an initial step during angiogenesis, in response to high-mobility group box-1 (HMGB1) that is released by cells during hypoxic injury. In vivo, ischemia-induced upregulation of TLR2 required intact TLR4 signaling that mediated systemic inflammation, as measured by local and systemic IL-6 levels. Similar to our in vitro findings, vascular density and limb perfusion were both enhanced in the absence of TLR4 signaling, but not if TLR2 was deleted. These findings indicate that TLR2, in the absence of TLR4, improves angiogenesis and perfusion recovery in response to ischemia.     

TLR4‐mediated skin carcinogenesis is dependent on immune and radioresistant cells

Skin cancers are the most commonly diagnosed cancers. Understanding what are the factors contributing to skin tumour development can be instrumental to identify preventive therapies. The myeloid differentiation primary response gene (MyD)88, the downstream adaptor protein of most Toll‐like receptors (TLR), has been shown to be involved in several mouse tumourigenesis models. We show here that TLR4, but not TLR2 or TLR9, is upstream of MyD88 in skin tumourigenesis. TLR4 triggering is not dependent on lipopolysaccharide associated to skin‐colonizing bacteria, but on the high mobility group box‐1 protein (HMGB1), an endogenous ligand of TLR4. HMGB1 is released by necrotic keratinocytes and is required for the recruitment of inflammatory cells and for the initiation of inflammation. The expression of TLR4 on both bone marrow‐derived and radioresistant cells is necessary for carcinogenesis. Consistently, a human tissue microarray analysis showed that melanoma and colon cancer display an over‐expression of TLR4 and its downstream adaptor protein MyD88 within tumours. Together, our results suggest that the initial release of HMGB1 triggers a TLR4‐dependent inflammatory response that leads to tumour development.     

低氧对人肺动脉平滑肌和人肺动脉内皮细胞中HMGB1及相关炎症因子表达的影响*

目的:探讨低氧时人肺动脉平滑肌细胞(HPASMC)和人肺动脉内皮细胞(HPAEC)的高迁移率族蛋白1(HMGB1)及相关受体和炎症因子表达,并检测HMGB1对两种细胞增殖、迁移活性的影响。方法:低氧(1%氧浓度,Hypoxia组)及常氧(Control组)条件下培养HPASMC和HPAEC,RealTime-PCR检测两种细胞HMGB1、TLR2、TLR4、TLR9、RAGE、CD24、IL-6 、TNF-a和CXCL8 mRNA等受体和炎性因子的表达。MTS法观察不同浓度HMGB1对HPASMC和HPAEC增殖的影响;划痕法观察HMGB1对HPASMC和HPAEC迁移的影响。结果:Hypoxia组HPASMC、HPAEC中HMGB1及RAGE mRNA表达量较Control 组明显升高(P＜0.05及0.01);Hypoxia组HPAEC中CD24及HPASMC中IL-6 mRNA表达明显增高(P均＜0.05)。MTS结果显示在345 pmol/L 剂量下 HMGB1明显抑制HPAEC的增殖(P＜0.01),而对HPASMC增殖无影响。划痕实验示HMGB1对HPASMC和HPAEC迁移无明显影响。结论:低氧诱导HPAEC、HPASMC 产生HMGB1;HMGB1通过抑制HPAEC增殖引起内皮屏障功能障碍;而低氧进一步刺激HPASMC产生炎症因子。     

高速泳动族蛋白1的致炎症作用机制

高速泳动族蛋白1(high-mobility group box 1,HMGB1)是一种高度保守的DNA结合蛋白,具有维持核小体结构和调节基因转录的功能,近来发现它是炎性反应强有力的促炎因子。在大多炎性疾病,特别是脓毒症病例中,HMGB1的血清和组织水平均显著升高,而且它与其受体如糖基化终末产物受体(receptor for advanced glycation end products,RAGE)、Toll样受体4(toll-like receptor,TLR4)、Toll样受体2(TLR2)等相互作用促进炎性疾病的发展。为了进一步了解HMGB1,本文就HMGB1的结构、生物学活性、与免疫细胞相互作用、细胞表面受体、以及拮抗HMGB1的药物等进行综述。     

A murine cellular model of necroinflammation displays RAGE‐dependent cytokine induction that connects to hepatoma cell injury

Unresolved inflammation maintained by release of danger‐associated molecular patterns, particularly high‐mobility group box‐1 (HMGB1), is crucial for hepatocellular carcinoma (HCC) pathogenesis. To further characterize interactions between leucocytes and necrotic cancerous tissue, a cellular model of necroinflammation was studied in which murine Raw 264.7 macrophages or primary splenocytes were exposed to necrotic lysates (N‐lys) of murine hepatoma cells or primary hepatocytes. In comparison to those derived from primary hepatocytes, N‐lys from hepatoma cells were highly active—inducing in macrophages efficient expression of inflammatory cytokines like C‐X‐C motif ligand‐2 , tumor necrosis factor‐α, interleukin (IL)‐6 and IL‐23‐p19. This activity associated with higher levels of HMGB1 in hepatoma cells and was curbed by pharmacological blockage of the receptor for advanced glycation end product (RAGE)/HMGB1 axis or the mitogen‐activated protein kinases ERK1/2 pathway. Analysis of murine splenocytes furthermore demonstrated that N‐lys did not comprise of functionally relevant amounts of TLR4 agonists. Finally, N‐lys derived from hepatoma cells supported inflammatory splenic Th17 and Th1 polarization as detected by IL‐17, IL‐22 or interferon‐γ production. Altogether, a straightforward applicable model was established which allows for biochemical characterization of immunoregulation by HCC necrosis in cell culture. Data presented indicate a remarkably inflammatory capacity of necrotic hepatoma cells that, at least partly, depends on the RAGE/HMGB1 axis and may shape immunological properties of the HCC microenvironment.     

Redox modification of cysteine residues regulates the cytokine activity of high mobility group box-1 (HMGB1)

High mobility group box 1 (HMGB1) is a nuclear protein with extracellular inflammatory cytokine activity. It is released passively during cell injury and necrosis, and secreted actively by immune cells. HMGB1 contains three conserved redox-sensitive cysteine residues: C23 and C45 can form an intramolecular disulfide bond, whereas C106 is unpaired and is essential for the interaction with Toll-Like Receptor (TLR) 4. However, a comprehensive characterization of the dynamic redox states of each cysteine residue and of their impacts on innate immune responses is lacking. Using tandem mass spectrometric analysis, we now have established that the C106 thiol and the C23-C45 disulfide bond are required for HMGB1 to induce nuclear NF-κB translocation and tumor necrosis factor (TNF) production in macrophages. Both irreversible oxidation to sulphonates and complete reduction to thiols of these cysteines inhibited TNF production markedly. In a proof of concept murine model of hepatic necrosis induced by acetaminophen, during inflammation, the predominant form of serum HMGB1 is the active one, containing a C106 thiol group and a disulfide bond between C23 and C45, whereas the inactive form of HMGB1, containing terminally oxidized cysteines, accumulates during inflammation resolution and hepatic regeneration. These results reveal critical posttranslational redox mechanisms that control the proinflammatory activity of HMGB1 and its inactivation during pathogenesis.     

Anti‐angiogenic effect of quercetin and its 8‐methyl pentamethyl ether derivative in human microvascular endothelial cells

Angiogenesis is involved in many pathological states such as progression of tumours, retinopathy of prematurity and diabetic retinopathy. The latter is a more complex diabetic complication in which neurodegeneration plays a significant role and a leading cause of blindness. The vascular endothelial growth factor (VEGF) is a powerful pro‐angiogenic factor that acts through three tyrosine kinase receptors (VEGFR‐1, VEGFR‐2 and VEGFR‐3). In this work we studied the anti‐angiogenic effect of quercetin (Q) and some of its derivates in human microvascular endothelial cells, as a blood retinal barrier model, after stimulation with VEGF‐A. We found that a permethylated form of Q, namely 8MQPM, more than the simple Q, is a potent inhibitor of angiogenesis both in vitro and ex vivo. Our results showed that these compounds inhibited cell viability and migration and disrupted the formation of microvessels in rabbit aortic ring. The addition of Q and more significantly 8MQPM caused recoveries or completely re‐establish the transendothelial electrical resistance (TEER) to the control values and suppressed the activation of VEGFR2 downstream signalling molecules such as AKT, extracellular signal‐regulated kinase, and c‐Jun N‐terminal kinase. Taken together, these data suggest that 8MQPM might have an important role in the contrast of angiogenesis‐related diseases.     

Diallyl trisulfide inhibits migration,invasion and angiogenesis of human colon cancer HT‐29 cells and umbilical vein endothelial cells,and suppresses murine xenograft tumour growth

Angiogenesis inhibitors are beneficial for the prevention and treatment of angiogenesis‐dependent diseases including cancer. We examined the cytotoxic, anti‐metastatic, anti‐cancer and anti‐angiogenic effects of diallyl trisulfide (DATS). In HT29 cells, DATS inhibited migration and invasion through the inhibition of focal adhesion kinase (FAK), extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase and p38 which was associated with inhibition of matrix metalloproteinases‐2, ‐7 and ‐9 and VEGF. In human umbilical vein endothelial cells (HUVEC), DATS inhibited the migration and angiogenesis through FAK, Src and Ras. DATS also inhibited the secretion of VEGF. The capillary‐like tube structure formation and migration by HUVEC was inhibited by DATS. The chicken egg chorioallantoic membrane (CAM) assay indicated that DATS treatment inhibited ex‐vivo angiogenesis. We investigated the anti‐tumour effects of DATS against human colon cancer xenografts in BALB/c^nu/nu mice and its anti‐angiogenic activity in vivo. In this in‐vivo study, DATS also inhibited the tumour growth, tumour weight and angiogenesis (decreased the levels of haemoglobin) in HT29 cells. In conclusion, the present results suggest that the inhibition of angiogenesis may be an important mechanism in colon cancer chemotherapy by DATS.