Expression Of Angiopoietin-1 Research Articles

Homeobox D9 drives the malignant phenotypes and enhances the Programmed death ligand-1 expression in non-small cell lung cancer cells via binding to Angiopoietin-2 promoter

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Homeobox D9 (HOXD9), a member of the HOX family of transcription factors, plays a driver role in development of multiple cancers. Angiopoietin-2 (ANGPT2) is reportedly to facilitate angiogenesis, growth and metastasis in various cancers, including lung cancer. In addition, blocking ANGPT2 can effectively improve cancer immunotherapy via downregulation of Programmed death ligand-1 (PD-L1). The purpose of this study was to elucidate the role of HOXD9 in NSCLC and whether ANGPT2 is required for HOXD9-mediated malignant behaviors of NSCLC cells. By performing a series of in vitro functional experiments, we found that knockdown of HOXD9 induced proliferative inhibition, cell cycle G1 arrest, apoptosis, migratory suppression and invasive repression of NSCLC cells. Reduced PD-L1 expression in NSCLC cells was observed after HOXD9 silencing. Besides, HOXD9 deletion decreased the expression of ANGPT2 in NSCLC cells. In line with this, HOXD9 overexpression led to opposite alteration in NSCLC cells. Mechanistically, ANGPT2 was transcriptionally activated by HOXD9. Forced expression of ANGPT2 significantly regulated HOXD9-mediated malignant phenotypes, and enhanced PD-L1 expression of NSCLC cells. Our results expressing HOXD9 may function as an oncogene in NSCLC via trans-activation of ANGPT2.

ANGPT2/CAV1 regulates albumin transcytosis of glomerular endothelial cells under high glucose exposure and is impaired by losartan

BackgroundMicroalbuminuria is a common clinical symptom that manifests in the early stages of diabetic kidney disease (DKD) and is also the main feature of glomerular endothelial cells (GECs) injury. There is increasing evidence that the transcytosis of albumin across GECs is closely related to the formation of albuminuria. Our previous studies have shown that angiopoietin 2 (ANGPT2) can inhibit albumin transcytosis across renal tubular epithelial cells by activating caveolin 1 (CAV1) phosphorylation during high glucose (HG) exposure. The role of ANGPT2 in albumin transcytosis across GECs remains unclear. Losartan significantly reduces albuminuria, but the mechanism has not been clarified. MethodsWe established an in vitro albumin transcytosis model to investigate the change in albumin transcytosis across human renal glomerular endothelial cells (hrGECs) under normal glucose (NG), high glucose (HG) and losartan intervention. We knocked down ANGPT2 and CAV1 to evaluate their roles in albumin transcytosis across hrGECs and verified the relationship between them. In vivo, DKD mouse models were established and treated with different doses of losartan. Immunohistochemistry and Western blot were used to detect the expression of ANGPT2 and CAV1. ResultsIn vitro, the transcytosis of albumin across hrGECs was significantly increased under high glucose stimulation, and losartan inhibited this process. The expression of ANGPT2 and CAV1 were both increased in hrGECs under HG conditions and losartan intervention reduced the expression of them. Moreover, ANGPT2 downregulation reduced albumin transcytosis in hrGECs by regulating CAV1 expression. In vivo, the expression of ANGPT2 and CAV1 in the glomerulus was both increased significantly in DKD mice. Compared with DKD mice, losartan treatment reduced albuminuria and decreased the expression of ANGPT2 and CAV1 in a dose-dependent manner. ConclusionsANGPT2 exacerbated albumin transcytosis across GECs by increasing CAV1 expression during HG exposure, thereby increasing albuminuria. Losartan reduces albumin transcytosis and albuminuria formation in DKD by inhibiting the upregulation of ANGPT2 under HG conditions. Our findings suggest that ANGPT2 and CAV1 may be novel therapeutic targets for diabetic albuminuria. In addition, we provide new evidence to elaborate on the mechanism of losartan in the development of DKD.

The effect of palm oil-derived tocotrienol-rich fraction in preserving normal retinal vascular diameter in streptozotocin-induced diabetic rats.

Angiogenesis in diabetic retinopathy (DR) is associated with increased retinal expression of angiopoietin-2 (Ang-2) and protein kinase C (PKC). Tocotrienol-rich fraction (TRF) has been shown to reduce the expression vascular endothelial growth factor (VEGF) in several experimental models. However, its effect against other angiogenic markers such as Ang-2 and PKC in rat modelof diabetes remains unknown. Therefore, we investigated the effect of TRF on the retinal vascular changes and Ang-2 and PKC expressions in rats with streptozotocin (STZ)-induced DR. Sprague-Dawley rats were divided into normal control rats (N) which received vehicle, and diabetic rats which either received vehicle (DV) or 100mg/kg of TRF (DT). Diabetes was induced with intraperitoneal injection of STZ (60mg/kg body weight). Treatments were given orally, once daily, for 12weeks after confirmation of hyperglycaemia. Fundus photographs were captured at baseline, 6- and 12-week post-STZ injection and average diameter of retinal veins and arteries were measured. At 12-week post-STZ injection, rats were euthanised, and retinae were collected for measurement of Ang-2 and PKC gene and protein expressions. Retinal venous and arterial diameters were significantly greater in DV compared to DT at week 12 post-STZ injection (p < 0.001 and < 0.05, respectively). The vesseldiameter measurements in DT were comparable to N and this effect of TRF was associated with significantly lower Ang-2 and PKC gene and protein expressions compared to DV. Oral TRF reduces the expression of retinal angiogenic markers and preserves the retinalvascular diameter of rats withSTZ-induced DR.

Astragalus propinquus schischkin and Salvia miltiorrhiza bunge promote angiogenesis to treat myocardial ischemia via Ang-1/Tie-2/FAK pathway.

Astragalus propinquus Schischkin and Salvia miltiorrhiza Bunge (AS) have been clinically used as adjunctive drugs in the treatment of myocardial ischemia (MI). However, the effect and mechanism of AS on MI have yet to be fully recognized. Here, we explored the cardioprotective effect of their combined use, and the mechanism of promoting angiogenesis through pericyte recruitment. Our data revealed that AS reduced MI and protects cardiac function. AS-treated MI mice exhibited reduced ST-segment displacement and repolarization time, increased ejection fraction, and less BNP and NT-proBNP expression. Pathological studies showed that, AS reduced the area of infarcted myocardium and slowed down the progress of cardiac remodelling and fibrosis. In addition, AS increased the content of platelet-derived growth factor receptors β (PDGFR-β), platelet endothelial cell adhesion molecule-1 (CD31) and angiogenesis-related proteins including vascular endothelial cadherin (VE-cadherin), Vascular Endothelial Growth Factor (VEGF) and transforming growth factor β (TGF-β). Moreover, these botanical drugs upregulated the expression of Angiopoietin-1 (Ang-1), phosphorylated angiopoietin-1 receptor (p-Tie-2), focal adhesion kinase (FAK) and growth factor receptor bound protein 7 (GRB7), indicating that the cardioprotection-related angiogenesis effect was related to pericyte recruitment, which may be through Ang-1/Tie-2/FAK pathway. In summary, AS can treat MI by protecting cardiac function, attenuating cardiac pathological changes, and hindering the progression of heart failure, which is related to angiogenesis after pericyte recruitment. Therefore, AS at a certain dose can be a promising treatment for MI with broad application prospects.

Expression of Angiopoietin 2 in ovarian follicles of crossbred Malabari goat

Angiopoietin-2 (Ang-2) is a local ovarian growth factor belonging to the angiopoietin/Tie (tyrosine kinase with Ig and EGF homology domains) signalling pathway, one of the main pathways involved in ovarian follicular angiogenesis. The main objective of this study is to assess the differential protein expression of Ang-2 in the developmental phases of ovarian follicles of crossbred Malabari goats employing an immunohistochemical technique. Ang-2 protein expression was found in all developmental phases of ovarian follicles, including primordial, primary, small, and large antral ovarian follicles. Ang-2 was found in the granulosa cells, theca interna cells, follicular fluid (granulosa cells in the FF), endothelial cells of capillaries and germinal epithelium of the goat ovaries. The Quantitative Method-Percentage Positivity of Ang-2 expression revealed that it was higher in the initial stages of ovarian follicular development and decreased with later stages of follicular development in cross-bred Malabari goats. Moreover, the protein expression of Ang2 was found to be higher in granulosa cells (GC) than in theca interna (TI) cells of antral follicles as evident from the higher staining intensity in GC cells compared to TI cells. It is therefore concluded that the Ang-2 protein expression in ovarian follicular cells decreased with an increase in follicle size and development and based on the combined scores for immunostaining (staining intensity and the number of positive staining cells), the Ang-2 was found to be expressed more in the granulosa cell layer compared to the theca interna cells of the antral follicle in crossbred Malabari goats.

Vascular density and expression of vascular endothelial growth factor and angiopoietin-1 using immunofluorescence technique in patients with metastatic renal cell carcinoma treated with first-line sunitinib

Vascular density and expression of vascular endothelial growth factor and angiopoietin-1 using immunofluorescence technique in patients with metastatic renal cell carcinoma treated with first-line sunitinib

Concentrated Growth Factor Promotes Wound Healing Potential of HaCaT Cells by Activating the RAS Signaling Pathway.

The aim of this study was to explore the effect of concentrated growth factor (CGF) on the wound healing potential of human epidermal cells (HaCaT) in vitro and in vivo. CGF was extracted from venous blood using the centrifugal separation method. The CGF-conditioned medium was prepared from CGF gel immersed in Dulbecco's Modified Eagle medium. Crystal violet staining and wound healing assay were used to evaluate the proliferation and migration of HaCaT cells, respectively. Lipopolysaccharide (LPS) was used to test the anti-inflammatory function of CGF. An ELISA kit was employed to detect the concentration of growth factors and interleukins in CGF medium. mRNA and protein levels of angiogenic biomarkers (Angiopoietin-1 (ANGPT-1), vascular endothelial growth factor-A (VEGF-A) and Angiopoietin-2 (ANGPT-2) ) were determined by quantitative polymerase chain reaction (qPCR) and Western blot, respectively. A dorsal excisional wound model was recruited to test the wound healing effect of CGF in mice. Three-day treatment of HaCaT cells with CGF significantly promoted cell proliferation, which was followed by an increase in Vascular Endothelial Growth Factor (VEGF) and Fibroblast Growth Factor (FGF) levels in the medium. Cytokines (IL-6, IL-8 and TNF-α) were increased in LPS-stimulated HaCaT cells after 3 days, and CGF slightly inhibited the mRNA expression of these cytokines. The RAS signaling pathway was activated upon CGF treatment. Both RAS knockdown and an inhibitor of RAS (zoledronic acid) could block the migration of HaCaT cells after CGF treatment. Protein expressions of CD31, ANGPT-1, and VEGF-A were up-regulated in a dose-dependent manner upon CGF exposure. The protein level of ANGPT-2 was down-regulated after CGF treatment. CGF could promote wound healing in vivo, as demonstrated using the full skin defect model in nude mice. CGF was shown to promote wound repair in vitro and in vivo. The RAS cell signaling pathway was responsible for CGF stimulating the wound healing potential of HaCaT cells.

ANGPT2/CAV1 regulates albumin transcytosis of glomerular endothelial cells under high glucose exposure and is impaired by losartan

BackgroundMicroalbuminuria is a common clinical symptom that manifests in the early stages of diabetic kidney disease (DKD) and is also the main feature of glomerular endothelial cells (GECs) injury. There is increasing evidence that the transcytosis of albumin across GECs is closely related to the formation of albuminuria. Our previous studies have shown that angiopoietin 2 (ANGPT2) can inhibit albumin transcytosis across renal tubular epithelial cells by activating caveolin 1 (CAV1) phosphorylation during high glucose (HG) exposure. The role of ANGPT2 in albumin transcytosis across GECs remains unclear. Losartan significantly reduces albuminuria, but the mechanism has not been clarified. MethodsWe established an in vitro albumin transcytosis model to investigate the change in albumin transcytosis across human renal glomerular endothelial cells (hrGECs) under normal glucose (NG), high glucose (HG) and losartan intervention. We knocked down ANGPT2 and CAV1 to evaluate their roles in albumin transcytosis across hrGECs and verified the relationship between them. In vivo, DKD mouse models were established and treated with different doses of losartan. Immunohistochemistry and Western blot were used to detect the expression of ANGPT2 and CAV1. ResultsIn vitro, the transcytosis of albumin across hrGECs was significantly increased under high glucose stimulation, and losartan inhibited this process. The expression of ANGPT2 and CAV1 were both increased in hrGECs under HG conditions and losartan intervention reduced the expression of them. Moreover, ANGPT2 downregulation reduced albumin transcytosis in hrGECs by regulating CAV1 expression. In vivo, the expression of ANGPT2 and CAV1 in the glomerulus was both increased significantly in DKD mice. Compared with DKD mice, losartan treatment reduced albuminuria and decreased the expression of ANGPT2 and CAV1 in a dose-dependent manner. ConclusionsANGPT2 exacerbated albumin transcytosis across GECs by increasing CAV1 expression during HG exposure, thereby increasing albuminuria. Losartan reduces albumin transcytosis and albuminuria formation in DKD by inhibiting the upregulation of ANGPT2 under HG conditions. Our findings suggest that ANGPT2 and CAV1 may be novel therapeutic targets for diabetic albuminuria. In addition, we provide new evidence to elaborate on the mechanism of losartan in the development of DKD.

Skin, Liver, and Kidney Interactions Contribute to Skin Dryness in Aging KK-Ay/Tajcl Mice

Type 2 diabetes is a lifestyle-related disease that affects people worldwide and is especially prevalent in the elderly. Many elderly people with diabetes also complain of dry skin; however, the relationship between aging and dry skin in type 2 diabetes is unknown. The purpose of this study was to examine the interaction between aging and dry skin using the specific pathogen-free KK-Ay/TaJcl type 2 diabetes mouse model. Skin dryness in this model increases with age and was evaluated at 10, 27, 40, and 50 weeks. We observed increased mast cell expression, increased histamine and matrix metalloproteinase-1 levels, and decreased collagen expression in the skin of aging KK-Ay/TaJcl mice. In addition, the increased expression of angiopoietin 2, interleukin-6, tumor necrosis factor-α, and endostatin in the blood indicated kidney damage in this model. Aging KK-Ay/TaJcl mice also showed fatty liver pathology, which led to increased reactive oxygen species in the blood and liver, as well as the increased expression of M1 macrophages in the liver. These results showed that dry skin is associated with skin, kidney, and liver interactions in an aging type 2 diabetes mouse model.

Inhibition of ANGPT2 activates autophagy during hypertrophic scar formation via PI3K/AKT/mTOR pathway

BackgroundHypertrophic scar (HS), a fibroproliferative disorder caused by aberrant wound healing following skin injuries such as burns, lacerations and surgery, is characterized by invasive proliferation of fibroblasts and excessive extracellular matrix (ECM) accumulation. The dysregulation of autophagy is the pathological basis of HS formation. Previously, angiopoietin-2 (ANGPT2) was found to be overexpressed in HS fibroblasts (HSFs) compared with normal skin fibroblasts. However, whether ANGPT2 participates in the process of HS formation and the potential molecular mechanisms are not clear. ObjectiveThis study is intended to figure out the role of ANGPT2 and ANGPT2-mediated autophagy during the development of HS. MethodsRT-qPCR was used to detect ANGPT2 expression in HS tissues and HSFs. HSFs were transfected with sh-ANGPT2 to knock down ANGPT2 expression and then treated with MHT1485, the mTOR agonist. The effects of sh-ANGPT2 or MHT1485 on the proliferation, migration, autophagy and ECM accumulation of HSFs were evaluated by CCK-8 assay, Transwell assay and western blotting. The expression of PI3K/Akt/mTOR pathway-related molecules (p-PI3K, p-Akt and p-mTOR) was assessed by western blotting. ResultsANGPT2 expression was markedly upregulated in HS tissues and HSFs. ANGPT2 knockdown decreased the expression of p-PI3K, p-Akt and p-mTOR. ANGPT2 knockdown activated autophagy and inhibited the proliferation, migration, and ECM accumulation of HSFs. Additionally, the treatment of MHT1485, the mTOR agonist, on ANGPT2-downregulated HSFs, partially reversed the influence of ANGPT2 knockdown on HSFs. Study limitationsThe study lacks the establishment of more stable in vivo animal models of HS for investigating the effects of ANGPT2 on HS formation in experimental animals. ConclusionsANGPT2 downregulation represses growth, migration, and ECM accumulation of HSFs via autophagy activation by suppressing the PI3K/Akt/mTOR pathway. Our study provides a novel potential therapeutic target for HS.

The molecular mechanisms underlying neutrophil infiltration in vessel co-opting colorectal cancer liver metastases.

Colorectal cancer liver metastases (CRCLMs) have two major histopathological growth patterns (HGPs): desmoplastic (DHGP) and replacement (RHGP). The DHGP tumours derive their vasculature by angiogenesis, while the RHGP tumours use vessel co-option. Various studies have associated RHGP tumours with an unfavourable prognosis, as well as high levels of resistance to anti-angiogenic agents and chemotherapy. Recently, we reported higher numbers of neutrophils in the tumour microenvironment (TME) of vessel co-opting tumours compared to their angiogenic counterparts. However, the molecular mechanisms underlying this phenotype are unclear. Herein, we suggested a positive correlation between the expression of angiopoietin-1 (Ang1) in the hepatocytes and the presence of neutrophils in vessel co-opting tumours. Importantly, upregulation of Ang1 in the hepatocytes is associated with the presence of runt-related transcription factor-1 (RUNX1) in the neighboring cancer cells in vitro and in vivo. Altogether, our data suggest the molecular mechanisms by which neutrophils are infiltrated in vessel co-opting CRCLM lesions. This finding may yield novel therapeutic strategies for CRCLM patients in future.

Endothelial angiopoietin-2 overexpression in explanted livers identifies subjects at higher risk of recurrence of hepatocellular carcinoma after liver transplantation.

BackgroundThough the precise criteria for accessing LT are consistently being applied, HCC recurrence (HCC-R_LT) still affects more than 15% of the patients. We analyzed the clinical, histopathological, and biological features of patients with HCC to identify the predictive factors associated with cancer recurrence and survival after LT.MethodsWe retrospectively analyzed 441 patients with HCC who underwent LT in our center. Overall, 70 (15.8%) of them developed HCC-R_LT. We matched them by age at transplant and etiology with 70 non-recurrent patients. A comparable cohort from the Liver Transplant Centre of Bologna served as validation. The clinical and biochemical characteristics and pre-LT criteria (Milan, Metroticket, Metroticket_AFP, and AFP model) were evaluated. Histological analysis and immunohistochemistry for angiopoietin-2 in the tumor and non-tumor tissue of explanted livers were performed. Patients’ follow-up was until death, last clinical evaluation, or 31 December 2021. In patients with HCC-R_LT, the date of diagnosis of recurrence and anatomical site has been reported; if a biopsy of recurrence was available, histologic and immunohistochemical analyses were also performed.ResultsPatients were followed up for a mean period of 62.7 ± 54.7 months (median, 39 months). A higher risk of HCC-R_LT was evident for factors related indirectly (AFP) or directly (endothelial angiopoietin-2, microvascular invasion) to biological HCC aggressiveness. In multivariate analysis, only angiopoietin-2 expression was independently associated with recurrence. Extremely high levels of endothelial angiopoietin-2 expression were also found in hepatic recurrence and all different metastatic locations. In univariate analysis, MELD, Metroticket_AFP Score, Edmondson–Steiner grade, microvascular invasion, and endothelial angiopoietin-2 were significantly related to survival. In multivariate analysis, angiopoietin-2 expression, Metroticket_AFP score, and MELD (in both training and validation cohorts) independently predicted mortality. In time-dependent area under receiver operating characteristic curve analysis, the endothelial angiopoietin-2 expression had the highest specificity and sensitivity for recurrence (AUC 0.922, 95% CI 0.876–0.962, p < 0.0001).ConclusionsEndothelial angiopoietin-2 expression is a powerful independent predictor of post-LT tumor recurrence and mortality, highlighting the fundamental role of tumor biology in defining the patients’ prognosis after liver transplantation. The great advantage of endothelial angiopoietin-2 is that it is evaluable in HCC biopsy before LT and could drive a patient’s priority on the waiting list.

IL-1β promotes A7r5 and HASMC migration and invasion via the p38-MAPK/Angpt-2 pathway

The migration, proliferation, and inflammatory factor secretion of vascular smooth muscle cells (VSMCs) are involved in the important pathological processes of several vascular occlusive diseases, including coronary atherosclerosis (CAS). Interleukin 1β(IL-1β), as a bioactive mediator of VSMC synthesis and secretion, can promote the pathological progress of CAS. In this study, we further explored the underlying molecular mechanisms by which IL-1β regulates VSMC migration, invasion. We pretreated A7r5 and HASMC with IL-1β for 24 h, and measured the expression of IL-1β, proliferating cell nuclear antigen (PCNA), cyclin D1, matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 2 (MMP9) in the cells by Western blotting. Cell migration and invasion ability were measured by Transwell and wound healing assays. Cell viability was measured by an MTT assay. We found that IL-1β upregulated the expression of proliferation-related proteins (PCNA and Cyclin D1) in A7r5 and HASMC, and induces the secretion of MMP2 and MMP9, promotes cell invasion and migration. In addition, in A7r5 and HASMCs treated with IL-1β, the expression of Angiopoietin-2 (Angpt-2) increased in a time-dependent manner, transfection with si-Angpt-2 suppressed cell migration and invasion, with downregulated MMP2 and MMP9 expression. Parallelly, we further found that the p38-MAPK pathway is activated in cells induced by IL-1β, p38-MAPK inhibitors can down-regulate the expression of Angpt-2. Collectively, these data demonstrated that IL-1β promotes A7r5 and HASMC migration and invasion via the p38-MAPK/Angpt-2 pathway.

Prognostic role of expression of angiogenesis markers in hepatocellular carcinoma: A bioinformatics analysis.

The expression of angiopoietin (ANGPT) 1, ANGPT2, vascular endothelial growth factor (VEGF) A, VEGFB, VEGFC, VEGFD, and placental growth factor (PGF) is significantly higher in tumor tissues than in normal tissues in both unpaired and paired hepatocellular carcinoma (HCC) samples. ANGPT2, VEGFB, VEGFC, and PGF are primarily involved in regulating the activation of the epithelial-mesenchymal transition pathway; ANGPT1 is primarily involved in regulating the activation of the RAS/mitogen-activated protein kinase and receptor tyrosine kinase (RTK) pathways; VEGFA is engaged in regulating the RTK activation pathway; and VEGFD is mainly involved in regulating the activation of the tuberous sclerosis protein/mammalian target of rapamycin pathway. There is a significant difference in overall survival between HCC patients with high and low expression of ANGPT2, PGF, VEGFA, and VEGFD. Disease free survival (DFS) is significantly shorter in HCC patients with high ANGPT2, PGF, and VEGFA expression than in those with low ANGPT2, PGF, and VEGFA expression.

Gene profiling in dorso-ventral patterning of mouse tongue development.

The tongue is a muscular fleshy organ in the oral cavity that is anatomically divided into the dorsal, ventral, anterior, and posterior part. The intricate tissue organisation and diverse origins of the tongue make it a complex organ of the oral cavity. To reveal the signalling molecules involved in the formation of the dorsal and ventral parts of the tongue through microarray analysis. Dorsal and ventral tongue tissues were isolated from embryonic day 14 mice by micro-dissection. RNA was extracted from the dorsal and ventral tongue tissues separately for microarray analysis. Microarray data were confirmed by quantitative reverse transcription polymerase chain reaction and whole-mount in situ hybridisation. Microarray analysis revealed expression of 33,793 genes. Of these, 931 genes were found to be equally expressed in both the dorsal and ventral parts of the tongue. On limiting the fold-change cut-off to over 1.5-fold, 725 genes were expressed over 1.5-fold in the ventral part and 1,672 in the dorsal part of the tongue. The qPCR and whole-mount in situ hybridisation revealed the expressions of angiopoietin 2 (Angpt2), fibroblast growth factor 18 (Fgf18), mesenchyme homeobox gene1 (Meox1), and SPARC-related modular calcium binding 2 (Smoc2) in the ventral part of the tongue. Numerous signalling molecules can be selected from our microarray results to examine their roles in tongue development and disease model systems. In the near future, the selection of candidate genes and their functional evaluations will be performed through loss- and gain-of-function mutation studies.

Glycocalyx heparan sulfate cleavage promotes endothelial cell angiopoietin-2 expression by impairing shear stress-related AMPK/FoxO1 signaling.

Angiopoietin-2 (Ang-2) is a key mediator of vascular disease during sepsis, and elevated plasma levels of Ang-2 are associated with organ injury scores and poor clinical outcomes. We have previously observed that biomarkers of endothelial glycocalyx (EG) damage correlate with plasma Ang-2 levels, suggesting a potential mechanistic linkage between EG injury and Ang-2 expression during states of systemic inflammation. However, the cell signaling mechanisms regulating Ang-2 expression following EG damage are unknown. In the current study, we determined the temporal associations between plasma heparan sulfate (HS) levels as a marker of EG erosion and plasma Ang-2 levels in children with sepsis and in mouse models of sepsis. Second, we evaluated the role of shear stress–mediated 5′-adenosine monophosphate–activated protein kinase (AMPK) signaling in Ang-2 expression following enzymatic HS cleavage from the surface of human primary lung microvascular endothelial cells (HLMVECs). We found that plasma HS levels peaked before plasma Ang-2 levels in children and mice with sepsis. Further, we discovered that impaired AMPK signaling contributed to increased Ang-2 expression following HS cleavage from flow-conditioned HLMVECs, establishing a paradigm by which Ang-2 may be upregulated during sepsis.

Akt1-dependent expression of angiopoietin 1 and 2 in vascular smooth muscle cells leads to vascular stabilization

Retinal angiogenesis was delayed in VSMC-specific Akt1-deficient mice (Akt1∆SMC) but not in Akt2∆SMC mice. The proliferation of ECs, recruitment of pericytes, and coverage of VSMCs to the endothelium were defective in Akt1∆SMC. The silencing of Akt1 in VSMCs led to the downregulation of angiopoietin 1 (Ang1) and the upregulation of Ang2. The activation of Notch3 in VSMCs was significantly reduced in the retinas of Akt1∆SMC mice. Silencing Akt1 suppressed the activation of Notch3. Moreover, the silencing of Notch3 downregulated Ang1, whereas the overexpression of Notch3 intracellular domain (NICD3) enhanced Ang1 expression. The nuclear localization and transcriptional activity of yes-associated protein (YAP) were affected by the expression level of Akt1. Silencing YAP downregulated Ang2 expression, whereas overexpression of YAP showed the opposite results. Ang1 antibody and Ang2 suppressed endothelial sprouting of wild-type aortic tissues, whereas the Ang2 antibody and Ang1 facilitated the endothelial sprouting of aortic tissues from Akt1∆SMC mice. Finally, severe hemorrhage was observed in Akt1∆SMC mice, which was further facilitated under streptozotocin (STZ)-induced diabetic conditions. Therefore, the Akt1-Notch3/YAP-Ang1/2 signaling cascade in VSMCs might play an essential role in the paracrine regulation of endothelial function.

HOXB5-activated ANGPT2 promotes the proliferation, migration, invasion and angiogenic effect of esophageal cancer cells via activating ERK/AKT signaling pathway.

Esophageal cancer, which is the eighth most common cancer worldwide, has a poor prognosis and high mortality rate. The present study was designed to investigate the proliferation, migration, invasion and angiogenic effect of the homeobox B5 (HOXB5)/angiopoietin-2 (ANGPT2) interplay in esophageal cancer. The relative expression of ANGPT2 and HOXB5 in esophageal cancer and the association between gene expression was evaluated using data from Gene Expression Profiling Interactive Analysis databases. Following transduction of short hairpin RNA-ANGPT2#1/2 plasmids, ANGPT2 was silenced. Viability, proliferation and invasion of esophageal cancer cells were assessed using CCK-8, 5-EdU, colony formation, wound healing and Transwell assays, respectively. Moreover, the transcriptional activity of ANGPT2 and angiogenesis were detected with luciferase reporter, chromatin immunoprecipitation (CH-IP) and tube formation assays. The results of the present study indicated that ANGPT2 was upregulated, both in esophageal cancer cell lines and tissue and there was an association between the ANGPT2 upregulation and the poor patient prognosis. In addition, ANGPT2 silencing suppressed esophageal cancer cell proliferation, migration, invasion and angiogenesis. The HOXB5 expression was also increased in esophageal cancer, and transcriptionally activated ANGPT2. Moreover, HOXB5 overexpression reversed the effects of ANGPT2 silencing in esophageal cancer cells. Furthermore, ANGPT2 silencing inactivated ERK/AKT signaling, whereas the HOXB5 overexpression blocked this effect. In conclusion, ANGPT2, which was transcriptionally activated by HOXB5, activated the ERK/AKT signaling pathway to promote proliferation, metastasis and angiogenesis of esophageal cancer cells.

A156 Differential Expression of Angiopoietin like -3,4 and 8 in Non-Alcoholic Fatty Liver Disease Improved Rats Following Sleeve Gastrectomy

A156 Differential Expression of Angiopoietin like -3,4 and 8 in Non-Alcoholic Fatty Liver Disease Improved Rats Following Sleeve Gastrectomy

SM22α cell-specific HIF stabilization mitigates hyperoxia-induced neonatal lung injury.

Though survival rates for preterm infants are improving, the incidence of chronic lung disease of infancy, or bronchopulmonary dysplasia (BPD), remains high. Histologically, BPD is characterized by larger and fewer alveoli. Hypoxia-inducible factors (HIFs) may be protective in the context of hyperoxia-induced lung injury, but the cell-specific effects of HIF expression in neonatal lung injury remain unknown. Thus, we sought to determine whether HIF stabilization in SM22α-expressing cells can limit hyperoxia-induced neonatal lung injury. We generated SM22α-specific HIF-1α-stabilized mice (SM22α-PHD1/2-/- mice) by cross-breeding SM22α-promotor-driven Cre recombinase mice with prolyl hydroxylase PHD1flox/flox and PHD2flox/flox mice. Neonatal mice were randomized to 21% O2 (normoxia) or 80% O2 (hyperoxia) exposure for 14 days. For the hyperoxia recovery studies, neonatal mice were recovered from normoxia for an additional 10 wk. SM22α-specific HIF-1α stabilization mitigated hyperoxia-induced lung injury and preserved microvessel density compared with control mice for both neonates and adults. In SM22α-PHD1/2-/- mice, pulmonary artery endothelial cells (PAECs) were more proliferative and pulmonary arteries expressed more collagen IV compared with control mice, even under hyperoxic conditions. Angiopoietin-2 (Ang2) mRNA expression in pulmonary artery smooth muscle cells (PASMC) was greater in SM22α-PHD1/2-/- compared with control mice in both normoxia and hyperoxia. Pulmonary endothelial cells (PECs) cocultured with PASMC isolated from SM22α-PHD1/2-/- mice formed more tubes and branches with greater tube length compared with PEC cocultured with PASMC isolated from SM22α-PHD1/2+/+ mice. Addition of Ang2 recombinant protein further augmented tube formation for both PHD1/2+/+ and PHD1/2-/- PASMC. Cell-specific deletion of PHD1 and 2 selectively increases HIF-1α expression in SM22α-expressing cells and protects neonatal lung development despite prolonged hyperoxia exposure. HIF stabilization in SM22α-expressing cells preserved endothelial cell proliferation, microvascular density, increased angiopoietin-2 expression, and lung structure, suggesting a role for cell-specific HIF-1α stabilization to prevent neonatal lung injury.