Inflammatory biomarkers, angiogenesis and lymphangiogenesis in epicardial adipose tissue correlate with coronary artery disease.

In this study, we explored the relationship between inflammatory adipokine levels and coronary artery disease (CAD). We collected subcutaneous adipose tissues(SAT), pericardial adipose tissues(PAT), and epicardial adipose tissues (EAT) and serum samples from 26 inpatients with CAD undergone coronary artery bypass grafting and 20 control inpatients without CAD. Serum inflammatory adipokines were measured by ELISA. Quantitative real-time PCR and western blot were used to measure gene and protein expression. Adipocyte morphology was assessed by H&E staining. Immunohistochemistry and immunofluorescence were used to measure endothelial and inflammatory markers. Serum pro- and anti-inflammatory adipokine levels were higher and lower, respectively, in the CAD group than those in the control group (P < 0.05). In CAD, the pro-inflammatory adipokine levels via ELISA in EAT and PAT were elevated. Pro-inflammatory adipokine mRNA expression was increased, while anti-inflammatory adipokine mRNA expression decreased, in CAD relative to NCAD in EAT and PAT rather than SAT. In EAT, adipocyte area and macrophage-specific staining were lower, while lymphatic vessel marker expression was higher in CAD. Additionally, the endothelial marker expression in EAT was higher than PAT in CAD. The three tissue types had different blood vessel amounts in CAD. The regulation and imbalance expression of the novel biomarkers, including inflammatory adipokine, macrophage infiltration, angiogenesis, and lymphangiogenesis in EAT and PAT, may be related to the pathogenesis of CAD. The serum levels of inflammatory adipokines may correlate to CAD, which requires large sample size studies to get further validation before clinic practice.

Method to Study the Role of Galectins in Angiogenesis In Vivo Using the Chick Chorioallantoic Membrane Assay.

Angiogenesis is a complex multi-step process involving various activities of endothelial cells. These activities are influenced in vivo by environmental conditions like interactions with other cell types and the microenvironment. Galectins play a role in several of these interactions and are therefore required for proper execution of in vivo angiogenesis. This chapter describes a method to study galectins during physiologic and pathophysiologic angiogenesis in vivo using the chicken chorioallantoic membrane (CAM) assay.

Untangling Galectin-Mediated Circuits that Control Hypoxia-Driven Angiogenesis.

Development of an aberrant vascular network is a hallmark of the multistep pathological process of tumor growth and metastasis. In response to hypoxia, several pro-angiogenic factors are synthesized to support vascularization programs required for cancer progression. Emerging data indicate the involvement of glycans and glycan-binding proteins as critical regulators of vascular circuits in health and disease. Galectins may be regulated by hypoxic conditions and control angiogenesis in different physiopathological settings. These ß-galactoside-binding proteins may promote sprouting angiogenesis by interacting with different glycosylated receptors and triggering distinct signaling pathways. Understanding the role of galectins in tumor neovascularization will contribute to the design of novel anti-angiogenic therapies aimed at complementing current anti-cancer modalities and overcoming resistance to these treatments. Here we describe selected strategies and methods used to study the role of hypoxia-regulated galectins in the regulation of blood vessel formation.

Hypoxic training upregulates mitochondrial turnover and angiogenesis of skeletal muscle in mice.

AIMS: Hypoxic training promotes human cardiopulmonary function and exercise performance efficiently, but the myocellular mechanism has been less studied. We aimed to examine the effects of hypoxic trainings on mitochondrial turnover and vascular remodeling of skeletal muscle. MAIN METHODS: C57BL/6 J mice were divided into control, hypoxic exposure, exercise training, "live high-train low" (LHTL), and "live low-train high" (LLTH) groups (n = 8/group). Western blot and immunohistochemistry were used to evaluate mitochondrial turnover of gastrocnemius and angiogenesis of quadriceps after six weeks interventions. KEY FINDINGS: Compared with control group, both LHTL and LLTH increased phosphorylation levels of p38 MAPK markedly (p < 0.05). LLTH also elevated PGC-1α protein expression significantly (p < 0.05). All interventions did not influence Bnip3 and Drp-1 proteins levels (p > 0.05), while LLTH enhanced Parkin and Mff protein contents significantly (p < 0.05). Immunohistochemical analysis showed both LHTL and LLTH promoted CD31 and VEGF expressions (p < 0.05). ATP content, citrate synthase activities of gastrocnemius were robustly elevated in LHTL and LLTH groups (p < 0.01). The exercise training increased Mff protein and ATP content in gastrocnemius as well as VEGF expression in quadriceps (p < 0.05). The hypoxic exposure also increased ATP content, citrate synthase, and ATP synthase activities in gastrocnemius as well as VEGF expression in quadriceps (p < 0.01). SIGNIFICANCE: Our results suggested that hypoxic trainings, especially LLTH, promoted mitochondrial turnover and angiogenesis of skeletal muscle, which may be an underlying mechanism of hypoxic training-induced exercise capacity.

Photothermal therapeutic potency of plasmonic silver nanoparticles for apoptosis and anti-angiogenesis in testosterone induced benign prostate hyperplasia in rats.

AIMS: In this study, we used a near-infrared laser (NIR) to increase the potency of silver nanoparticles (AgNPs) to develop a novel, less invasive, and simple photothermal therapy technique for benign prostate hyperplasia (BPH). MATERIALS AND METHODS: The shape, particle size, and zeta-potential of polyvinylpyrrolidone coated-AgNPs (PVP-AgNPs) were determined using transmission electron microscopy (TEM), Zeta-potential, and Particle size analyzer (ELSZ). To induce BPH, thirty-six male Sprague-Dawley (SD) rats were given intramuscular (i.m) injections of testosterone propionate (TP) at 5 mg/kg body weight (b.w)/day suspended in 0.1 ml of olive oil for 14 days. Photothermal therapy with AgNPs-NIR for 14 days was carried out. Prostate size, prostate index (PI), dihydrotestosterone (DHT), prostate-specific antigen (PSA), gross, hepatic, and renal toxicity, as well as antioxidant activity, apoptosis, and angiogenesis markers in prostatic tissues were measured. Histological examinations of prostates and biocompatibility of NIR-AgNPs on vital organs were also performed. KEY FINDINGS: The aggregated spherical AgNPs with a mean size of 50-90 nm and a Zeta potential of -53.22 mV displayed high effectiveness in the NIR (532 nm-1 W) region by decreasing prostate size, PI, DHT, and PSA in BPH rats with no signs of gross, hepatic, or renal damage. As compared to alternative therapies, hyperthermia therapy increased antioxidant activities, induced apoptosis, inhibited angiogenesis, reduced histological alterations in the prostates of BPH rats, and improved biocompatibility of the vital organs. SIGNIFICANCE: The current study demonstrated the effectiveness of plasmonic AgNPs photothermal therapy in the treatment of BPH.

Impact of macular fluid features on outcomes of anti-vascular endothelial growth factor treatment for type 3 macular neovascularization.

We evaluated the impact of macular fluid features on visual and anatomical outcomes in type 3 macular neovascularization (MNV) patients treated with anti-vascular endothelial growth factor (VEGF). We retrospectively enrolled 89 eyes with type 3 MNV with at least 12 months of follow-up. All patients were treatment-naïve and received a monthly loading injection of anti-VEGF for three months, followed by further injections as required. The association of baseline macular morphology, including intraretinal fluid (IRF) and subretinal fluid (SRF), with visual and anatomical outcomes was analyzed. At baseline, IRF was present in all enrolled patients (100%), and SRF was present in 43.8% (39/89) of them. After 12 months of treatment, no significant difference was found in terms of best-corrected visual acuity (BCVA) and changes in central foveal thickness between the eyes with (39) and without (50) SRF at baseline. In addition, the proportion of improved or worsened (gain or loss of more than three lines in the BCVA) visual acuity at 12 months was not significantly different among the groups. Incidence of macular atrophy during the treatment showed no difference between the groups, regardless of the presence of SRF. In conclusion, the macular fluid morphology, specifically SRF, in type 3 MNV showed no significant correlation with visual and anatomical outcomes during anti-VEGF treatment.

The impact of chronic mild hypoxia on cerebrovascular remodelling; uncoupling of angiogenesis and vascular breakdown.

BACKGROUND: Chronic mild hypoxia (CMH, 8% O2) stimulates robust vascular remodelling in the brain, but it also triggers transient vascular disruption. This raises the fundamental question: is the vascular leak an unwanted side-effect of angiogenic remodelling or is it a pathological response, unrelated to endothelial proliferation, in which declining oxygen levels trigger endothelial dysfunction? METHODS: To answer this question, mice were exposed to CMH (8% O2) for periods up to 14 days, after which, brain tissue was examined by immunofluorescence (IF) to determine which type of blood vessel (arteriole, capillary or venule) was most commonly associated with endothelial proliferation and vascular leak and how this correlated with tight junction protein expression. Vascular perfusion was examined using DiI. Data were analysed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison post-hoc test. RESULTS: The following was observed: (1) most endothelial proliferation and extravascular fibrinogen leak occurred in capillaries and to a lesser degree in venules, (2) much to our surprise, endothelial proliferation and extravascular fibrinogen leak never colocalized, (3) interestingly however, endothelial proliferation was strongly associated with an intravascular fibrinogen staining pattern not seen in stable blood vessels, (4) DiI perfusion studies revealed that angiogenic vessels were adequately perfused, suggesting that fibrinogen retention in angiogenic vessels is not due to temporary closure of the vessel, but more likely because fibrinogen is retained within the vessel wall, (5) bromodeoxyuridine (BrdU) labelling as a means to more permanently label proliferating endothelial cells, confirmed lack of any connection between endothelial proliferation and extravascular fibrinogen leak, while (6) in contrast, proliferating microglia were detected within extravascular leaks. CONCLUSIONS: Taken together, our findings support the concept that in the short-term, hypoxia-induced endothelial proliferation triggers transient fibrinogen deposition within the walls of angiogenic blood vessels, but no overt vascular leak occurs in these vessels. Importantly, endothelial proliferation and extravascular fibrinogen leaks never co-localize, demonstrating that extravascular leak is not an unwanted side-effect of angiogenic endothelial proliferation, but rather a dysfunctional vascular response to hypoxia that occurs in a distinct group of non-angiogenic blood vessels.

Angiogenesis in Chronic Inflammatory Skin Disorders.

Angiogenesis, the growth of new blood vessels from preexisting vessels, is associated with inflammation in various pathological conditions. Well-known angiogenetic factors include vascular endothelial growth factor (VEGF), angiopoietins, platelet-derived growth factor, transforming growth factor-ß, and basic fibroblast growth factor. Yes-associated protein 1 (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) have recently been added to an important angiogenic factor. Accumulating evidence indicates associations between angiogenesis and chronic inflammatory skin diseases. Angiogenesis is deeply involved in the pathogenesis of psoriasis. VEGF, angiopoietins, tumor necrosis factor-a, interleukin-8, and interleukin-17 are unregulated in psoriasis and induce angiogenesis. Angiogenesis may be involved in the pathogenesis of atopic dermatitis, and in particular, mast cells are a major source of VEGF expression. Angiogenesis is an essential process in rosacea, which is induced by LL-37 from a signal cascade by microorganisms, VEGF, and MMP-3 from mast cells. In addition, angiogenesis by increased VEGF has been reported in chronic urticaria and hidradenitis suppurativa. The finding that VEGF is expressed in inflammatory skin lesions indicates that inhibition of angiogenesis is a useful strategy for treatment of chronic, inflammatory skin disorders.

Tumor-Associated Microglia/Macrophages as a Predictor for Survival in Glioblastoma and Temozolomide-Induced Changes in CXCR2 Signaling with New Resistance Overcoming Strategy by Combination Therapy.

Tumor recurrence is the main challenge in glioblastoma (GBM) treatment. Gold standard therapy temozolomide (TMZ) is known to induce upregulation of IL8/CXCL2/CXCR2 signaling that promotes tumor progression and angiogenesis. Our aim was to verify the alterations on this signaling pathway in human GBM recurrence and to investigate the impact of TMZ in particular. Furthermore, a combi-therapy of TMZ and CXCR2 antagonization was established to assess the efficacy and tolerability. First, we analyzed 76 matched primary and recurrent GBM samples with regard to various histological aspects with a focus on the role of TMZ treatment and the assessment of predictors of overall survival (OS). Second, the combi-therapy with TMZ and CXCR2-antagonization was evaluated in a syngeneic mouse tumor model with in-depth immunohistological investigations and subsequent gene expression analyses. We observed a significantly decreased infiltration of tumor-associated microglia/macrophages (TAM) in recurrent tumors, while a high TAM infiltration in primary tumors was associated with a reduced OS. Additionally, more patients expressed IL8 in recurrent tumors and TMZ therapy maintained CXCL2 expression. In mice, enhanced anti-tumoral effects were observed after combi-therapy. In conclusion, high TAM infiltration predicts a survival disadvantage, supporting findings of the tumor-promoting phenotype of TAMs. Furthermore, the combination therapy seemed to be promising to overcome CXCR2-mediated resistance.

ADAM9 functions as a transcriptional regulator to drive angiogenesis in esophageal squamous cell carcinoma.

Hypoxia and angiogenesis play key roles in the pathogenesis of esophageal squamous cell carcinoma (ESCC), but regulators linking these two pathways to drive tumor progression remain elusive. Here we provide evidence of ADAM9's novel function in ESCC progression. Increasing expression of ADAM9 was correlated with poor clinical outcomes in ESCC patients. Suppression of ADAM9 function diminished ESCC cell migration and in vivo metastasis in ESCC xenograft mouse models. Using cellular fractionation and imaging, we found a fraction of ADAM9 was present in the nucleus and was uniquely associated with gene loci known to be linked to the angiogenesis pathway demonstrated by genome-wide ChIP-seq. Mechanistically, nuclear ADAM9, triggered by hypoxia-induced translocation, functions as a transcriptional repressor by binding to promoters of genes involved in the negative regulation of angiogenesis, and thereby promotes tumor angiogenesis in plasminogen/plasmin pathway. Moreover, ADAM9 suppresses plasminogen activator inhibitor-1 gene transcription by interacting with its transcription factors at the promoter. Our findings uncover a novel regulatory mechanism of ADAM9 as a transcriptional regulator in angiogenesis and highlight ADAM9 as a promising therapeutic target for ESCC treatment.

Targeting the Angiopoietin/Tie Pathway: Prospects for Treatment of Retinal and Respiratory Disorders.

Anti-angiogenic approaches have significantly advanced the treatment of vascular-related pathologies. The ephemeral outcome and known side effects of the current vascular endothelial growth factor (VEGF)-based anti-angiogenic treatments have intensified research on other growth factors. The angiopoietin/Tie (Ang/Tie) family has an established role in vascular physiology and regulates angiogenesis, vascular permeability, and inflammatory responses. The Ang/Tie family consists of angiopoietins 1-4, their receptors, tie1 and 2 and the vascular endothelial-protein tyrosine phosphatase (VE-PTP). Modulation of Tie2 activation has provided a promising outcome in preclinical models and has led to clinical trials of Ang/Tie-targeting drug candidates for retinal disorders. Although less is known about the role of Ang/Tie in pulmonary disorders, several studies have revealed great potential of the Ang/Tie family members as drug targets for pulmonary vascular disorders as well. In this review, we summarize the functions of the Ang/Tie pathway in retinal and pulmonary vascular physiology and relevant disorders and highlight promising drug candidates targeting this pathway currently being or expected to be under clinical evaluation for retinal and pulmonary vascular disorders.

Endothelial Colony-Forming Cells Dysfunctions Are Associated with Arterial Hypertension in a Rat Model of Intrauterine Growth Restriction.

Infants born after intrauterine growth restriction (IUGR) are at risk of developing arterial hypertension at adulthood. The endothelium plays a major role in the pathogenesis of hypertension. Endothelial colony-forming cells (ECFCs), critical circulating components of the endothelium, are involved in vasculo-and angiogenesis and in endothelium repair. We previously described impaired functionality of ECFCs in cord blood of low-birth-weight newborns. However, whether early ECFC alterations persist thereafter and could be associated with hypertension in individuals born after IUGR remains unknown. A rat model of IUGR was induced by a maternal low-protein diet during gestation versus a control (CTRL) diet. In six-month-old offspring, only IUGR males have increased systolic blood pressure (tail-cuff plethysmography) and microvascular rarefaction (immunofluorescence). ECFCs isolated from bone marrow of IUGR versus CTRL males displayed a decreased proportion of CD31+ versus CD146+ staining on CD45- cells, CD34 expression (flow cytometry, immunofluorescence), reduced proliferation (BrdU incorporation), and an impaired capacity to form capillary-like structures (Matrigel test), associated with an impaired angiogenic profile (immunofluorescence). These dysfunctions were associated with oxidative stress (increased superoxide anion levels (fluorescent dye), decreased superoxide dismutase protein expression, increased DNA damage (immunofluorescence), and stress-induced premature senescence (SIPS; increased beta-galactosidase activity, increased p16INK4a, and decreased sirtuin-1 protein expression). This study demonstrated an impaired functionality of ECFCs at adulthood associated with arterial hypertension in individuals born after IUGR.

The development of tumour vascular networks.

The growth of solid tumours relies on an ever-increasing supply of oxygen and nutrients that are delivered via vascular networks. Tumour vasculature includes endothelial cell lined angiogenesis and the less common cancer cell lined vasculogenic mimicry (VM). To study and compare the development of vascular networks formed during angiogenesis and VM (represented here by breast cancer and pancreatic cancer cell lines) a number of in vitro assays were utilised. From live cell imaging, we performed a large-scale automated extraction of network parameters and identified properties not previously reported. We show that for both angiogenesis and VM, the characteristic network path length reduces over time; however, only endothelial cells increase network clustering coefficients thus maintaining small-world network properties as they develop. When compared to angiogenesis, the VM network efficiency is improved by decreasing the number of edges and vertices, and also by increasing edge length. Furthermore, our results demonstrate that angiogenic and VM networks appear to display similar properties to road traffic networks and are also subject to the well-known Braess paradox. This quantitative measurement framework opens up new avenues to potentially evaluate the impact of anti-cancer drugs and anti-vascular therapies.

The endothelium-bone axis in development, homeostasis and bone and joint disease.

Blood vessels form a versatile transport network that is best known for its critical roles in processes such as tissue oxygenation, metabolism and immune surveillance. The vasculature also provides local, often organ-specific, molecular signals that control the behaviour of other cell types in their vicinity during development, homeostasis and regeneration, and also in disease processes. In the skeletal system, the local vasculature is actively involved in both bone formation and resorption. In addition, blood vessels participate in inflammatory processes and contribute to the pathogenesis of diseases that affect the joints, such as rheumatoid arthritis and osteoarthritis. This Review summarizes the current understanding of the architecture, angiogenic growth and functional properties of the bone vasculature. The effects of ageing and pathological conditions, including arthritis and osteoporosis, are also discussed.

GPCR Partners as Cancer Driver Genes: Association with PH-Signal Proteins in a Distinctive Signaling Network.

The essential role of G-protein coupled receptors (GPCRs) in tumor growth is recognized, yet a GPCR based drug in cancer is rare. Understanding the molecular path of a tumor driver gene may lead to the design and development of an effective drug. For example, in members of protease-activated receptor (PAR) family (e.g., PAR1 and PAR2), a novel PH-binding motif is allocated as critical for tumor growth. Animal models have indicated the generation of large tumors in the presence of PAR1 or PAR2 oncogenes. These tumors showed effective inhibition when the PH-binding motif was either modified or were inhibited by a specific inhibitor targeted to the PH-binding motif. In the second part of the review we discuss several aspects of some cardinal GPCRs in tumor angiogenesis.

Analysis of the Internal Hypoxic Environment in Solid Tumor Tissue Using a Folding Paper System.

Hypoxia is a nonphysiological oxygen tension which is common in most malignant tumors. Hypoxia stimulates complicated cell signaling networks in cancer cells, e.g., the HIF, PI3K, MAPK, and NFκB pathways. Then, cells release a number of cytokines such as VEGFA to promote the growth of peripheral blood vessels and lead to metastasis. In the current work, understanding of the internal hypoxic environment in solid tumor tissue was attempted by developing a folding paper system. A paper-based solid tumor was constructed by folding a filter paper cultured with cancer cells. The cellular response in each layer could be analyzed by disassembling the folded paper after the culture course. The result showed that an internal hypoxic environment was successfully reproduced in the paper-based solid tumor. The cells in the inner layer expressed high levels of HIF1-α and VEGFA. Hence, proliferation and migration of endothelial cells were shown to be induced by the cells located in the internal hypoxic environment. Moreover, the paper-based solid tumor was transplanted into nude mice for the study of hypoxic response and angiogenesis. The crosstalk between internal and external parts of solid tumor tissue could be analyzed by sectioning each layer of the paper-based solid tumor. This approach provides a favorable analytical method for the discovery of the interaction between cancer cells, hypoxia, and peripheral angiogenesis.

Exosomes and their cargo are important regulators of cell function in endometriosis.

Endometriosis is a chronic oestrogen-dependent gynaecological disorder characterized by non-menstrual pelvic pain, infertility and the extrauterine growth of endometrial-like glands and stroma. It has been noted that the eutopic endometrium of women with endometriosis is functionally distinct from that of women without endometriosis. Moreover, ectopic endometrial implants are functionally different from the eutopic endometrium of women with endometriosis. However, the mechanisms directing these differences are ill-defined. It is proposed here that small membrane-bound extracellular vesicles called exosomes are important vehicles in the protection and transport of signalling molecules central to the dysregulation of endometrial function in women with endometriosis. Therefore, a critical review of the literature linking exosomes and their cargo to the pathobiology of endometriosis was conducted. Circulating peritoneal fluid and endometrial cell exosomes contained long non-coding RNA, miRNA and proteins involved in histone modification, angiogenesis and immune modulation that differed significantly in women with endometriosis compared with controls. Moreover, experimental evidence supports a role for exosomes and their cargo in angiogenesis, neurogenesis, immune modulation and endometrial stromal cell invasion. It is therefore suggested that exosomes play an important role in the pathophysiology of endometriosis.

The mechanisms of renin-angiotensin system in hepatocellular carcinoma: From the perspective of liver fibrosis, HCC cell proliferation, metastasis and angiogenesis, and corresponding protection measures.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, of which the occurrence and development involve a variety of pathophysiological processes, such as liver fibrosis, hepatocellular malignant proliferation, metastasis, and tumor angiogenesis. Some important cytokines, such as TGF-ß, PI3K, protein kinase B (Akt), VEGF and NF-κB, can regulate the growth, proliferation, diffusion, metastasis, and apoptosis of HCC cells by acting on the corresponding signaling pathways. Besides, many studies have shown that the formation of HCC is closely related to the main components of renin-angiotensin system (RAS), such as Ang II, ACE, ACE2, MasR, AT1R, and AT2R. Therefore, this review focused on liver fibrosis, HCC cell proliferation, metastasis, tumor angiogenesis, and corresponding protective measures. ACE-Ang II-AT1 axis and ACE2-Ang-(1-7)-MasR axis were taken as the main lines to introduce the mechanism of RAS in the occurrence and development of HCC, so as to provide references for future clinical work and scientific research.

Unaffected fellow eye neovascularization in patients with type 3 neovascularization: Incidence and risk factors.

PURPOSE: To evaluate the incidence and risk factors of neovascularization in unaffected fellow eyes of patients diagnosed with type 3 neovascularization in Korea. METHODS: This retrospective study included 93 unaffected fellow eyes of 93 patients diagnosed with type 3 neovascularization. For initial type 3 neovascularization diagnosis, optical coherence tomography and angiography were conducted. These baseline data were compared between patients with and without neovascularization in their fellow eyes during the follow-up period. RESULTS: The mean follow-up period was 66.1±31.1 months. Neovascularization developed in 49 (52.8%) fellow eyes after a mean period of 29.5±19.6 months. In the fellow eye neovascularization group, the incidence of soft drusen and reticular pseudodrusen was significantly higher than that in the non-neovascularization group (83.7% vs. 36.5%, p<0.001; 67.3% vs. 40.9%, p = 0.017, respectively), but the choroidal vascularity index (CVI) showed a significantly lower value (60.7±2.0% vs. 61.7±2.5%; p = 0.047). The presence of reticular pseudodrusen was related with the duration from baseline to development of fellow eye neovascularization (p = 0.038). CONCLUSION: Neovascularization developed in 52.8% of unaffected fellow eyes. The presence of soft drusen, reticular pseudodrusen, and lower CVI values can be considered risk factors of neovascularization in unaffected fellow eyes of patients with type 3 neovascularization. The lower CVI values suggest that choroidal ischemic change may affect the development of choroidal neovascularization in these patients.