Hypoxia-mediated Angiogenesis Research Articles

TRIM2 Selectively Regulates Inflammation-Driven Pathological Angiogenesis without Affecting Physiological Hypoxia-Mediated Angiogenesis.

Angiogenesis is a critical physiological response to ischemia but becomes pathological when dysregulated and driven excessively by inflammation. We recently identified a novel angiogenic role for tripartite-motif-containing protein 2 (TRIM2) whereby lentiviral shRNA-mediated TRIM2 knockdown impaired endothelial angiogenic functions in vitro. This study sought to determine whether these effects could be translated in vivo and to determine the molecular mechanisms involved. CRISPR/Cas9-generated Trim2-/- mice that underwent a periarterial collar model of inflammation-induced angiogenesis exhibited significantly less adventitial macrophage infiltration relative to wildtype (WT) littermates, concomitant with decreased mRNA expression of macrophage marker Cd68 and reduced adventitial proliferating neovessels. Mechanistically, TRIM2 knockdown in endothelial cells in vitro attenuated inflammation-driven induction of critical angiogenic mediators, including nuclear HIF-1α, and curbed the phosphorylation of downstream effector eNOS. Conversely, in a hindlimb ischemia model of hypoxia-mediated angiogenesis, there were no differences in blood flow reperfusion to the ischemic hindlimbs of Trim2-/- and WT mice despite a decrease in proliferating neovessels and arterioles. TRIM2 knockdown in vitro attenuated hypoxia-driven induction of nuclear HIF-1α but had no further downstream effects on other angiogenic proteins. Our study has implications for understanding the role of TRIM2 in the regulation of angiogenesis in both pathophysiological contexts.

Extracellular Vesicles from Adipose-Derived Stem Cells Promote Diabetic Wound Healing via the PI3K-AKT-mTOR-HIF-1α Signaling Pathway.

Impaired potential of hypoxia-mediated angiogenesis lead poor healing of diabetic wounds. Previous studies have shown that extracellular vesicles from adipose derived stem cells (ADSC-EVs) accelerate wound healing with unelucidated mechanism. However, it is not yet clear about the underlying mechanism of ADSC-EVs in regulating the hypoxia-related PI3K/AKT/mTOR signaling pathway of vascular endothelial cells in diabetic wounds. Therefore, in this study, human derived ADSC-EVs (hADSC-EVs) isolated from adipose tissue were co-cultured with advanced glycosylation end product (AGE) treated human umbilical vein endothelial cells (HUVECs) in vitro and local injected into the wounds of diabetic rats. In vitro, the therapeutic potential of hADSC-EVs on AGE-treated HUVECs was evaluated by cell counting kit-8, scratching, and tube formation assay. Subsequently, the effects of hADSC-EVs on the PI3K/AKT/mTOR/HIF-1α signaling pathway were also assayed by qRT-PCR and western blot. In vivo, the effect of hADSC-EVs on diabetic wound healing in rats were also assayed by closure kinetics, Masson staining and HIF-1α-CD31 immunofluorescence. hADSC-EVs were spherical in shape with an average particle size of 198.1 ± 91.5nm, and were positive for CD63, CD9 and TSG101. hADSC-EVs promoted the expression of PI3K-AKT-mTOR-HIF-1α signaling pathway of AGEs treated HUVECs with improved the potential of proliferation, migration and tube formation, and improve the healing and angiogenesis of diabetic wound in rats. However, the effect of hADSC-EVs described above can be blocked by PI3K-AKT inhibitor both in vitro and vivo. Our findings indicated that hADSC-EVs accolated the healing of diabetic wounds by promoting HIF-1α-mediated angiogenesis in the PI3K-AKT-mTOR depend manner.

HIF2-Induced Long Noncoding RNA RAB11B-AS1 Promotes Hypoxia-Mediated Angiogenesis and Breast Cancer Metastasis.

Hypoxia induces a vast array of long noncoding RNAs (lncRNA) in breast cancer cells, but their biological functions remain largely unknown. Here, we identified a hitherto uncharacterized hypoxia-induced lncRNA RAB11B-AS1 in breast cancer cells. RAB11B-AS1 is a natural lncRNA upregulated in human breast cancer and its expression is induced by hypoxia-inducible factor 2 (HIF2), but not HIF1, in response to hypoxia. RAB11B-AS1 enhanced the expression of angiogenic factors including VEGFA and ANGPTL4 in hypoxic breast cancer cells by increasing recruitment of RNA polymerase II. In line with increased angiogenic factors, conditioned media from RAB11B-AS1-overexpressing breast cancer cells promoted tube formation of human umbilical vein endothelial cells in vitro. Gain- and loss-of-function studies revealed that RAB11B-AS1 increased breast cancer cell migration and invasion in vitro and promoted tumor angiogenesis and breast cancer distant metastasis without affecting primary tumor growth in mice. Taken together, these findings uncover a fundamental mechanism of hypoxia-induced tumor angiogenesis and breast cancer metastasis. SIGNIFICANCE: This study reveals the molecular mechanism by which the lncRNA RAB11B-AS1 regulates hypoxia-induced angiogenesis and breast cancer metastasis, and provides new insights into the functional interaction between a lncRNA and tumor microenvironment. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/5/964/F1.large.jpg.

003 TRIM2 is a Novel Inhibitor of Inflammatory-Driven Pathological Angiogenesis, but Preserves Physiological Hypoxia-Mediated Angiogenesis

003 TRIM2 is a Novel Inhibitor of Inflammatory-Driven Pathological Angiogenesis, but Preserves Physiological Hypoxia-Mediated Angiogenesis

A lncRNA coordinates with Ezh2 to inhibit HIF-1α transcription and suppress cancer cell adaption to hypoxia.

Hypoxia is a salient feature of the tumor microenvironment. HIF-1α is a master regulator of hypoxic adaption. The polycomb repressor complex 2 (PRC2) molecule Ezh2 is known to play roles in essential cellular processes of cell fate decisions. However, how PRC2-mediated epigenetic dynamic changes take part in hypoxic adaption is not completely understood. Recently, we identified a long non-coding RNA (lncRNA) named HITT (HIF-1α inhibitor at translation levels) that plays roles in modulating hypoxia-mediated angiogenesis and tumor growth in vivo. In this study, we reveal an important activity of HITT in evading hypoxia-induced apoptosis by coordinating with PRC2 activity to regulate HIF-1α transcription. Genetic or chemical inhibition of PRC2 significantly elevates HIF-1α mRNA levels. The occupancy of Ezh2 and its substrate H3K27me3 on the HIF-1α promoter is detected under normoxia, and is reduced by hypoxia. Restoring hypoxia-inhibited HITT expression rescues the association between Ezh2/H3K27me3 and the HIF-1α promoter, which also simultaneously abrogates hypoxia-induced HIF-1α mRNA transcription. Further mechanistic studies revealed that HITT inhibits HIF-1α transcription by guiding Ezh2 through the formation of an RNA-DNA triplex with the HIF-1α promoter. Importantly, HITT/Ezh2-regulated HIF-1α transcription leads to alerted HIF-1α protein output and elicits a significant effect to evade hypoxia-induced apoptosis. Importantly, a close association between HIF-1α mRNA and HITT was further verified in human colon cancer tissues in vivo. Collectively, these findings suggest a model for the epigenetic regulation of hypoxia-induced HIF-1α transcription modulated by lncRNA HITT, which provides important insights into how tumor cells sense and adapt to hypoxic stress.

Noninvasive induction of angiogenesis in tissues by external suction: sequential optimization for use in reconstructive surgery.

In reconstructive surgery, tissues are routinely transferred to repair a defect caused by trauma, cancer, chronic diseases, or congenital malformations; surgical transfer intrinsically impairs metabolic supply to tissues placing a risk of ischemia-related complications such as necrosis, impaired healing, or infection. Pre-surgical induction of angiogenesis in tissues (preconditioning) can limit postsurgical ischemic complications and improve outcomes, but very few preconditioning strategies have successfully been translated to clinical practice due to the invasiveness of most proposed approaches, their suboptimal effects, and their challenging regulatory approval. We optimized a method that adopts noninvasive external suction to precondition tissues through the induction of hypoxia-mediated angiogenesis. Using a sequential approach in a rodent model, we determined the parameters of application (frequency, suction levels, duration, and interfaces) that fine-tune the balance of enhanced angiogenesis, attenuation of hypoxic tissue damage, and length of treatment. The optimized repeated short-intermittent applications of intermediate suction induced a 1.7-fold increase in tissue vascular density after only 5days of treatment (p<0.05); foam interfaces showed the same effectiveness and caused less complications. In a second separate experiment, our model showed that the optimized technique significantly improves survival of transferred tissues. Here we demonstrate that noninvasive external suction can successfully, safely, and promptly enhance vascularity of soft tissues: these translational principles can help design effective preconditioning strategies, transform best clinical practice in surgery, and improve patient outcomes.

Strontium- and cobalt-substituted bioactive glasses seeded with human umbilical cord perivascular cells to promote bone regeneration via enhanced osteogenic and angiogenic activities.

Bone regeneration is considered as an unmet clinical need. The most recent researches focused on incorporation of strontium (Sr2+) and cobalt (Co2+) ions into bioactive glasses structure. Strontium is an alkaline earth metal which is currently used in the treatment of osteoporosis. Also, cobalt is considered as another promising element in the bone regeneration field that may induce hypoxia-mediated angiogenesis. In this study, the osteogenic potential of the strontium- and cobalt-substituted bioactive glasses in granule form seeded with human umbilical cord perivascular cells (HUCPVCs) was evaluated in vitro and in vivo. Indeed, the main goal of this study was to improve the osteogenenic and angiogenic properties of bioactive glasses through the incorporation of strontium and cobalt ions in the glass composition. Although some researches have been conducted on this subject, the influence of the simultaneous use of strontium and cobalt ions on the improvement of bone healing in vivo has been not yet well understood and, therefore, deserves further investigation.

The Emerging Role of Histone Demethylases in Renal Cell Carcinoma.

Renal cell carcinoma (RCC), the most common kidney cancer, is responsible for more than 100,000 deaths per year worldwide. The molecular mechanism of RCC is poorly understood. Many studies have indicated that epigenetic changes such as DNA methylation, noncoding RNAs, and histone modifications are central to the pathogenesis of cancer. Histone demethylases (KDMs) play a central role in histone modifications. There is emerging evidence that KDMs such as KDM3A, KDM5C, KDM6A, and KDM6B play important roles in RCC. The available literature suggests that KDMs could promote RCC development and progression via hypoxia-mediated angiogenesis pathways. Small-molecule inhibitors of KDMs are being developed and used in preclinical studies; however, their clinical relevance is yet to be established. In this mini review, we summarize our current knowledge on the putative role of histone demethylases in RCC.

CC-chemokine class inhibition attenuates pathological angiogenesis while preserving physiological angiogenesis.

Increasing evidence shows that CC-chemokines promote inflammatory-driven angiogenesis, with little to no effect on hypoxia-mediated angiogenesis. Inhibition of the CC-chemokine class may therefore affect angiogenesis differently depending on the pathophysiological context. We compared the effect of CC-chemokine inhibition in inflammatory and physiological conditions. In vitro, the broad-spectrum CC-chemokine inhibitor "35K" inhibited inflammatory-induced endothelial cell proliferation, migration, and tubulogenesis, with more modest effects in hypoxia. In vivo, adenoviruses were used to overexpress 35K (Ad35K) and GFP (AdGFP, control virus). Plasma chemokine activity was suppressed by Ad35K in both models. In the periarterial femoral cuff model of inflammatory-driven angiogenesis, overexpression of 35K inhibited adventitial neovessel formation compared with control AdGFP-infused mice. In contrast, 35K preserved neovascularization in the hindlimb ischemia model and had no effect on physiological neovascularization in the chick chorioallantoic membrane assay. Mechanistically, 2 key angiogenic proteins (VEGF and hypoxia-inducible factor-1α) were conditionally regulated by 35K, such that expression was inhibited in inflammation but was unchanged in hypoxia. In conclusion, CC-chemokine inhibition by 35K suppresses inflammatory-driven angiogenesis while preserving physiological ischemia-mediated angiogenesis via conditional regulation of VEGF and hypoxia-inducible factor-1α. CC-chemokine inhibition may be an alternative therapeutic strategy for suppressing diseases associated with inflammatory angiogenesis without inducing the side effects caused by global inhibition.- Ridiandries, A., Tan, J. T. M., Ravindran, D., Williams, H., Medbury, H. J., Lindsay, L., Hawkins, C., Prosser, H. C. G., Bursill, C. A. CC-chemokine class inhibition attenuates pathological angiogenesis while preserving physiological angiogenesis.

Multiple across-strain and within-strain QTLs suggest highly complex genetic architecture for hypoxia tolerance in channel catfish

The ability to survive hypoxic conditions is important for various organisms, especially for aquatic animals. Teleost fish, representing more than 50% of vertebrate species, are extremely efficient in utilizing low levels of dissolved oxygen in water. However, huge variations exist among various taxa of fish in their ability to tolerate hypoxia. In aquaculture, hypoxia tolerance is among the most important traits because hypoxia can cause major economic losses. Genetic enhancement for hypoxia tolerance in catfish is of great interest, but little was done with analysis of the genetic architecture of hypoxia tolerance. The objective of this study was to conduct a genome-wide association study to identify QTLs for hypoxia tolerance using the catfish 250K SNP array with channel catfish families from six strains. Multiple significant and suggestive QTLs were identified across and within strains. One significant QTL and four suggestive QTLs were identified across strains. Six significant QTLs and many suggestive QTLs were identified within strains. There were rare overlaps among the QTLs identified within the six strains, suggesting a complex genetic architecture of hypoxia tolerance. Overall, within-strain QTLs explained larger proportion of phenotypic variation than across-strain QTLs. Many of genes within these identified QTLs have known functions for regulation of oxygen metabolism and involvement in hypoxia responses. Pathway analysis indicated that most of these genes were involved in MAPK or PI3K/AKT/mTOR signaling pathways that were known to be important for hypoxia-mediated angiogenesis, cell proliferation, apoptosis and survival.

Suppression of Akt-HIF-1α signaling axis by diacetyl atractylodiol inhibits hypoxia-induced angiogenesis.

Hypoxia-inducible factor (HIF)-1α is a key regulator associated with tumorigenesis, angiogenesis, and metastasis. HIF-1α regulation under hypoxia has been highlighted as a promising therapeutic target in angiogenesis-related diseases. Here, we demonstrate that diacetyl atractylodiol (DAA) from Atractylodes japonica (A. japonica) is a potent HIF-1α inhibitor that inhibits the Akt signaling pathway. DAA dose-dependently inhibited hypoxia-induced HIF-1α and downregulated Akt signaling without affecting the stability of HIF-1α protein. Furthermore, DAA prevented hypoxia-mediated angiogenesis based on in vitro tube formation and in vivo chorioallantoic membrane (CAM) assays. Therefore, DAA might be useful for treatment of hypoxia-related tumorigenesis, including angiogenesis. [BMB Reports 2016; 49(9): 508-513]

MiR675-5p Acts on HIF-1α to Sustain Hypoxic Responses: A New Therapeutic Strategy for Glioma.

Hypoxia is a common feature in solid tumours. In glioma, it is considered the major driving force for tumour angiogenesis and correlates with enhanced resistance to conventional therapies, increased invasiveness and a poor prognosis for patients. Here we describe, for the first time, that miR675-5p, embedded in hypoxia-induced long non-coding RNA H19, plays a mandatory role in establishing a hypoxic response and in promoting hypoxia-mediated angiogenesis. We demonstrated, in vitro and in vivo, that miR675-5p over expression in normoxia is sufficient to induce a hypoxic moreover, miR675-5p depletion in low oxygen conditions, drastically abolishes hypoxic responses including angiogenesis. In addition, our data indicate an interaction of miR675-5p, HIF-1α mRNA and the RNA Binding Protein HuR in hypoxia-induced responses. We suggest the modulation of miR675-5p as a new therapeutic option to promote or abolish hypoxia induced angiogenesis.

Arginase inhibition enhances angiogenesis in endothelial cells exposed to hypoxia

Hypoxia-induced arginase elevation plays an essential role in several vascular diseases but influence of arginase on hypoxia-mediated angiogenesis is completely unknown. In this study, in vitro network formation in bovine aortic endothelial cells (BAEC) was examined after exposure to hypoxia for 24h with or without arginase inhibition. Arginase activity, protein levels of the two arginase isoforms, eNOS, and VEGF as well as production of NO and ROS were examined to determine the involvement of arginase in hypoxia-mediated angiogenesis. Hypoxia elevated arginase activity and arginase 2 expression but reduced active p-eNOSSer1177 and NO levels in BAEC. In addition, both VEGF protein levels and endothelial elongation and network formation were reduced with continued hypoxia, whereas ROS levels increased and NO levels decreased. Arginase inhibition limited ROS, restored NO formation and VEGF expression, and prevented the reduction of angiogenesis. These results suggest a fundamental role of arginase activity in regulating angiogenic function.

Inhibition of dual specificity phosphatase-2 by hypoxia promotes interleukin-8-mediated angiogenesis in endometriosis.

How does hypoxia-mediated down-regulation of dual specificity phosphatase-2 (DUSP2) promote endometriotic lesion development? Inhibition of DUSP2 by hypoxia enhances endometriotic lesion growth via promoting interleukin-8 (IL-8)-dependent angiogenesis. Angiogenesis is a prerequisite for the development of endometriosis. DUSP2 is down-regulated in endometriotic stromal cells in a hypoxia inducible factor-1α-dependent manner. Down-regulation of DUSP2 contributes to the pathological process of endometriosis. A laboratory study recruiting 20 patients of reproductive age with endometriosis and normal menstrual cycles, and an autoimplant-induced mouse model of endometriosis using 13 mice in a 28-day treatment. IL-8 mRNA levels were assayed in endometrial stromal cells maintained in normoxic or hypoxic (1% O2) conditions, with or without DUSP2 knockdown. Promoter activity and chromatin immunoprecipitation (ChIP) assays were conducted to characterize the regulation of IL-8 by DUSP2. Conditioned media from cells maintained in normoxic or hypoxic conditions, and cells with/without DUSP2 knockdown were collected to investigate the angiogenic capacity using an in vitro tube formation assay. Reparixin, an IL-8 receptor blocker, was administered to investigate the role of IL-8 in hypoxia-mediated angiogenesis and the development of endometriotic-like lesions in an autotransplanted mouse model. IL-8 mRNA was increased by both hypoxia and DUSP2 knockdown in endometrial stromal cells in an extracellular signal-regulated protein kinase-dependent manner (P < 0.05 versus control). Promoter activity and ChIP assays demonstrated that expression of IL-8 was regulated by CCAAT/enhancer binding protein α (P < 0.05 versus control). Furthermore, conditioned media collected from hypoxia-exposed or DUSP2 knockdown endometrial stromal cells promoted tube formation, which was abolished by co-treatment with reparixin (P < 0.05 versus control). Results from the autotransplanted mouse model demonstrated that number of blood vessels and size of endometriotic-like lesions were markedly reduced in recipient mice treated with reparixin (P < 0.05 versus control). This study was conducted in primary human cell cultures and a mouse model, therefore may not fully reflect the situation in vivo. This is the first study to highlight the potential application of an IL-8 receptor blocker as a therapeutic target to treat endometriosis. This study demonstrates IL-8 as a key angiogenic factor regulated by hypoxia/DUSP2, which suggests an alternative mechanism through which hypoxia may promote angiogenesis. This study was funded by the National Science Council of Taiwan (NSC101-2314-B-006-043-MY2). The author declares that there is no conflict of interest.

Multifunctional regulation of angiogenesis by high-density lipoproteins

High-density lipoproteins (HDL) exert striking anti-inflammatory effects and emerging evidence suggests that they may augment ischaemia-mediated neovascularization. We sought to determine whether HDL conditionally regulates angiogenesis, depending on the pathophysiological context by (i) inhibiting inflammation-induced angiogenesis, but also; (ii) enhancing ischaemia-mediated angiogenesis. Intravenously delivered apolipoprotein (apo) A-I attenuated neovascularization in the murine femoral collar model of inflammation-induced angiogenesis, compared with phosphate-buffered saline infused C57BL6/J mice (58%), P < 0.05. Conversely, apoA-I delivery augmented neovessel formation (75%) and enhanced blood perfusion (45%) in the murine hindlimb ischaemia model, P < 0.05. Reconstituted HDL (rHDL) was tested on key angiogenic cell functions in vitro. rHDL inhibited human coronary artery endothelial cell migration (37.9 and 76.9%), proliferation (15.7 and 40.4%), and tubulogenesis on matrigel (52 and 98.7%) when exposed to two inflammatory stimuli: tumour necrosis factor-α (TNF-α) and macrophage-conditioned media (MCM). In contrast, rHDL significantly augmented hypoxia-stimulated migration (36.9%), proliferation (135%), and tubulogenesis (22.9%), P < 0.05. Western blot and RT-PCR analyses revealed that these divergent actions of rHDL were associated with conditional regulation of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and VEGF receptor 2, which were attenuated in response to TNF-α (40.4, 41.0, and 33.2%) and MCM (72.5, 30.7, and 69.5%), but augmented by rHDL in hypoxia (39.8, 152.6, and 15.7%%), all P < 0.05. HDL differentially regulates angiogenesis dependent upon the pathophysiological setting, characterized by suppression of inflammation-associated angiogenesis, and conversely, by the enhancement of hypoxia-mediated angiogenesis. This has significant implications for therapeutic modulation of neovascularization.

RUNX3 inhibits hypoxia-inducible factor-1α protein stability by interacting with prolyl hydroxylases in gastric cancer cells

RUNX3 is silenced by histone modification and hypoxia-inducible factor (HIF)-1α is stabilized under hypoxia, but little is known of cross-talk between RUNX3 and HIF-1α under hypoxia. In the present study, the authors investigated the effect of RUNX3 on HIF-1α stability in gastric cancer cells. RUNX3 overexpression was found to downregulate HIF-1α stability under normoxic and hypoxic conditions. Furthermore, the activity of a luciferase reporter containing five copies of vascular endothelial growth factor (VEGF) promoter hypoxia-responsive element (5 × HRE) and the amount of secreted VEGF, were diminished in RUNX3-expressing but increased in RUNX3-knockdown cells. When expression of RUNX3 was recovered using epigenetic reagents the expressions of HIF-1α and VEGF were clearly suppressed under hypoxic conditions. RUNX3 also significantly attenuated the half-life of HIF-1α protein, and induced the cytosolic localization and ubiquitination of HIF-1α. In addition, RUNX3 directly interacted with the C-terminal activation domain of HIF-1α and prolyl hydroxylase (PHD) 2 and enhanced the interaction between HIF-1α and PHD2, which potentiated proline hydroxylation and promoted the degradation of HIF-1α. Furthermore, RUNX3 overexpression significantly inhibited hypoxia-induced angiogenesis in vitro and in vivo. Taken together, these results suggest that RUNX3 destabilizes HIF-1α protein by promoting the proline hydroxylation of HIF-1α through binding to HIF-1α/PHD2. RUNX3 appears to be a novel suppressor of HIF-1α and of hypoxia-mediated angiogenesis in gastric cancer cells.

Abstract 392: High-Density Lipoproteins Beneficially Regulate Angiogenesis

Introduction: Angiogenesis can be both favourable and unfavourable. It is critical for collateral vessel formation in response to ischaemia following myocardial infarction (MI), however excessive inflammatory-driven neovascularisation accelerates atherosclerotic plaque growth. High-density lipoproteins (HDL) are associated with improved survival and prognosis following MI as well as reduced plaque development. Hypothesis: We hypothesized that HDL may beneficially regulate angiogenesis under both hypoxic or inflammatory settings, in vitro. Methods and results: Human coronary artery endothelial cells (HCAECs) subjected to hypoxia (1.2% O2, 6h) displayed increased tubulogenesis (19%) and proliferation (31%), p&lt;0.05. Interestingly, pretreatment with reconstituted HDL (rHDL), containing apolipoprotein (apo)A-I and phosphatidylcholine, augmented hypoxia-driven elevations in tubulogenesis (14%) and proliferation (53%), p&lt;0.05. Consistent with this, rHDL also augmented hypoxia-driven elevations in the proangiogenic mediators HIF1α (39%), VEGF (1.5 fold) and SDFα (3.4 fold), p&lt;0.05. In contrast to the findings in hypoxia, pre-treatment with rHDL followed by exposure to two inflammatory conditions: TNFα (0.7 ng/ml) and stimulated macrophage-conditioned media (CM), significantly inhibited HCAEC tubulogenesis (75% &amp; 70%), proliferation (47% &amp; 39%) and migration (37% &amp; 55%), p&lt;0.05. The anti-angiogenic effects of rHDL in both inflammatory settings were associated with reductions in the pro-angiogenic inflammatory mediators p65 (43% &amp; 52%) and MCP-1 (33% &amp; 30%), p&lt;0.05. Moreover, in contrast to the setting of hypoxia, rHDL reduced HIF1α and VEGF protein in response to inflammation (TNFα (36% &amp; 34%) and CM (37% &amp; 19%), p&lt;0.05). rHDL pretreatment strikingly increased hemeoxygenase-1 (HO-1) protein expression, a known regulator of angiogenesis, under both hypoxic and inflammatory conditions (∼2-4 fold for all), p&lt;0.05. Conclusions: In summary, we have identified HDL to uniquely regulate angiogenesis, characterized by enhancement of hypoxia-mediated angiogenesis and, conversely, marked suppression of inflammation-associated angiogenesis.

Multifunctional Regulation of Angiogenesis by High Density Lipoproteins

Introduction: Angiogenesis is a key biological process in both physiological and pathological states. It is critical for neovascularisation in response to ischaemia; however, excessive inflammatory-driven angiogenesis accelerates atherosclerotic plaque growth. High-density lipoproteins (HDL) are associated with improved prognosis following myocardial infarction, and with reduced atherogenesis. We hypothesised that HDL may regulate angiogenesis in a multifunctional manner: by augmenting hypoxia-mediated angiogenesis while inhibiting inflammation-driven angiogenesis. Methods and results: Treatment of human coronary artery endothelial cells (ECs) with reconstituted HDL (rHDL, 600 μg/mL) augmented hypoxia (1%O2, 6 h)-mediated stimulation of EC proliferation (53%) and tubulogenesis in matrigel (14%) (p < 0.05). These rHDL-mediated effects were associated with augmentation of hypoxia-related induction of hypoxia inducible factor-1α (HIF1α) (39%) by downregulation of prolyl hydroxylase 2 & 3 (30% & 52%) and induction of Siah 1 & 2 (36% & 60%), post-translational regulators of HIF1α stability. Consistent with HIF1α induction, rHDL stimulated EC protein expression of the key proangiogenic factors vascular endothelial growth factor (VEGF) (50%) and stromal cell-derived factor-1α in hypoxia (p < 0.05). In contrast to hypoxia, rHDL significantly inhibited EC tubulogenesis (75% & 70%), proliferation (47% & 39%) and migration (37% & 55%), following exposure to two inflammatory stimuli: tumour necrosis factor-α (TNFα) and macrophage-conditioned media (CM) (p < 0.05). The anti-angiogenic effects of rHDL in inflammation were associated with reduced pro-angiogenic inflammatory mediators: p65 (43% & 52%) and monocyte chemoattractant protein (33% & 30%) (p < 0.05). Strikingly, under inflammation, rHDL reduced HIF1α and VEGF protein expression: 36% & 34% (TNFα), 37% & 19% (CM) (p < 0.05). rHDL increased hemeoxygenase-1 expression, a multi-functional regulator of angiogenesis, under both hypoxic & inflammatory conditions (p < 0.05). Conclusions: HDL uniquely regulates angiogenesis in a multifunctional manner, characterised by enhancement of hypoxia-mediated angiogenesis and, conversely, marked suppression of inflammation-associated angiogenesis.

Abstract 3092: FBW7 targets hypoxia inducible factor-1α (HIF-1α) for proteasomal degradation during hypoxia

Abstract Hypoxia Inducible Factor-1α (HIF-1α) mediates expression of genes associated with endothelial cell-mediated angiogenesis and is associated with poor outcomes in a variety of cancers. In normoxia, HIF-1α is ubiquitinated and degraded through interactions with the E3 ubiqutin ligase, von Hippel-Lindau (vHL); however, little is known about the negative regulation of HIF-1α in hypoxia. FBW7, an E3 ubiquitin ligase, has been shown to interact with several transcription factors including those phosphorylated by glycogen synthase kinase 3β (GSK3β). The current study tested the hypothesis that phosphorylation of HIF-1α by GSK3β increases the FBW7-mediated ubiquitination and degradation of HIF-1α, thereby resulting in suppression of the hypoxia-mediated angiogenic response in SKOV-3 ovarian cancer cells. HIF-1α protein and VEGF transcript levels in hypoxia were increased when GSK3β activity was inhibited and were reduced by expression of constitutively active GSK3β (GSK3S9A). Additionally, expression of GSK3S9A increased HIF-1α ubiquitination. Conditioned media from SKOV-3 cells expressing shRNA against GSK3β (shGSK3) had an enhanced effect on endothelial tube formation in a Matrigel matrix, suggesting that GSK3β exerts an inhibitory effect on hypoxia-mediated angiogenesis. Overexpression of the β and γ isoforms of FBW7 decreased HIF-1α stabilization, and HIF-1α interacts with FBW7 by co-immunoprecipitation. Furthermore, knockdown of FBW7 using siRNA resulted in increased HIF-1α levels in hypoxia. These data suggest a new mechanism for negative regulation of HIF-1α during hypoxia that utilizes phosphorylation by GSK3β and interaction with FBW7 leading to ubiquitination and proteasomal degradation. Results of this study better define the signaling pathways necessary for HIF-1α-mediated signaling and may identify new targets that mediate angiogenesis in disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3092. doi:10.1158/1538-7445.AM2011-3092

Inhibitory action of Celastrol on hypoxia-mediated angiogenesis and metastasis via the HIF-1α pathway

Celastrol, a natural biologically active compound isolated from Tripterygium wilfordii Hook F root extracts, has been shown to possess antitumor properties and therefore, is an interesting candidate for the development of novel chemotherapeutic cancer agents. In this study, we have demonstrated that Celastrol is a potent inhibitor of hypoxia-induced angiogenic and metastatic activity as shown by a decrease in the proliferation of both endothelial and cancer cells, blocking of migration as well as of tube formation of endothelial cells, and by inhibition of cancer cell invasion under hypoxic conditions. Moreover, Celastrol decreased hypoxia-inducible factor-1α (HIF-1α) mRNA levels under both normoxia and hypoxia and inhibited hypoxia-induced accumulation of nuclear HIF-1α protein. Meanwhile, inhibition of nuclear HIF-1α protein levels were accompanied by a reduction in the transcriptional activity of HIF-1α target genes, including VEGF. In addition, the inhibitory effect of Celastrol on HIF-1α protein was partly due to its suppression of HSP90 activity. We conclude that Celastrol regulates HIF-1α at multiple levels that may together or individually contribute to its antitumor activity against hypoxia-induced angiogenesis and metastasis.