Recurrence After Atrial Fibrillation Ablation and Investigational Biomarkers of Cardiac Remodeling.

BACKGROUND: Recurrence after atrial fibrillation (AF) ablation remains common. We evaluated the association between recurrence and levels of biomarkers of cardiac remodeling, and their ability to improve recurrence prediction when added to a clinical prediction model. METHODS AND RESULTS: Blood samples collected before de novo catheter ablation were analyzed. Levels of bone morphogenetic protein-10, angiopoietin-2, fibroblast growth factor-23, insulin-like growth factor-binding protein-7, myosin-binding protein C3, growth differentiation factor-15, interleukin-6, N-terminal pro-brain natriuretic peptide, and high-sensitivity troponin T were measured. Recurrence was defined as ≥30 seconds of an atrial arrhythmia 3 to 12 months postablation. Multivariable logistic regression was performed using biomarker levels along with clinical covariates: APPLE score (Age >65 years, Persistent AF, imPaired eGFR [<60 ml/min/1.73m2], LA diameter ≥43 mm, EF <50%; which includes age, left atrial diameter, left ventricular ejection fraction, persistent atrial fibrillation, and estimated glomerular filtration rate), preablation rhythm, sex, height, body mass index, presence of an implanted continuous monitor, year of ablation, and additional linear ablation. A total of 1873 participants were included. A multivariable logistic regression showed an association between recurrence and levels of angiopoietin-2 (odds ratio, 1.08 [95% CI, 1.02-1.15], P=0.007) and interleukin-6 (odds ratio, 1.02 [95% CI, 1.003-1.03]; P=0.02). The area under the receiver operating characteristic curve of a model that only contained clinical predictors was 0.711. The addition of any of the 9 studied biomarkers to the predictive model did not result in a statistically significant improvement in the area under the receiver operating characteristic curve. CONCLUSIONS: Higher angiopoietin-2 and interleukin-6 levels were associated with recurrence after atrial fibrillation ablation in multivariable modeling. However, the addition of biomarkers to a clinical prediction model did not significantly improve recurrence prediction.

How we use angiopoietin-2 in the diagnosis and management of vascular anomalies.

The diagnosis of vascular anomalies remains challenging due to significant clinical heterogeneity and uncertain etiology. Evaluation using biopsy and/or genetic testing for somatic variants is invasive, expensive, and prone to sampling error. There is great need for noninvasive and easily measured blood laboratory biomarkers that can aid not only in diagnosis, but also management of treatments for vascular anomalies. Angiopoietin-2, a circulating blood angiogenic factor, is highly elevated in patients with kaposiform hemangioendothelioma with Kasabach-Merritt phenomenon and kaposiform lymphangiomatosis. Here, we describe our clinical experience using serum angiopoietin-2 as a biomarker for diagnosis and monitoring response to treatment.

CPAP may promote an endothelial inflammatory milieu in sleep apnoea after coronary revascularization.

BACKGROUND: Continuous positive airway pressure (CPAP) has failed to reduce cardiovascular risk in obstructive sleep apnoea (OSA) in randomized trials. CPAP increases angiopoietin-2, a lung distension-responsive endothelial proinflammatory marker associated with increased cardiovascular risk. We investigated whether CPAP has unanticipated proinflammatory effects in patients with OSA and cardiovascular disease. METHODS: Patients with OSA (apnoea-hypopnea index [AHI] ≥15 events/h without excessive sleepiness) in the Randomized Intervention with CPAP in Coronary Artery Disease and OSA study were randomized to CPAP or usual care following coronary revascularization. Changes in plasma levels of biomarkers of endothelial (angiopoietin-2, Tie-2, E-selectin, vascular endothelial growth factor [VEGF-A]) and lung epithelial (soluble receptor of advanced glycation end-products [sRAGE]) function from baseline to 12-month follow-up were compared across groups and associations with cardiovascular morbidity and mortality assessed. FINDINGS: Patients with OSA (n = 189; 84% men; age 66 ± 8 years, BMI 28 ± 3.5 kg/m2, AHI 41 ± 23 events/h) and 91 patients without OSA participated. Angiopoietin-2 remained elevated whereas VEGF-A declined significantly over 12 months in the CPAP group (n = 91). In contrast, angiopoietin-2 significantly declined whereas VEGF-A remained elevated in the usual care (n = 98) and OSA-free groups. The changes in angiopoietin-2 and VEGF-A were significantly different between CPAP and usual care, whereas Tie-2, sRAGE and E-selectin were similar. Greater 12-month levels of angiopoietin-2 were associated with greater mortality. Greater CPAP levels were associated with worse cardiovascular outcomes. INTERPRETATION: Greater CPAP levels increase proinflammatory, lung distension-responsive angiopoietin-2 and reduce cardioprotective angiogenic factor VEGF-A compared to usual care, which may counteract the expected cardiovascular benefits of treating OSA. FUNDING: National Institutes of Health/National Heart, Lung, and Blood Institute; Swedish Research Council; Swedish Heart-Lung Foundation; ResMed Foundation.

Elevated CSF angiopoietin-2 correlates with blood-brain barrier leakiness and markers of neuronal injury in early Alzheimer's disease.

Breakdown of the neurovascular unit is associated with blood-brain barrier (BBB) leakiness contributing to cognitive decline and disease pathology in the early stages of Alzheimer's disease (AD). Vascular stability depends on angiopoietin-1 (ANGPT-1) signalling, antagonised by angiopoietin-2 (ANGPT-2) expressed upon endothelial injury. We examined the relationship between CSF ANGPT-2 and CSF markers of BBB leakiness and core AD biomarkers across three independent cohorts: (i) 31 AD patients and 33 healthy controls grouped according to their biomarker profile (i.e., AD cases t-tau > 400 pg/mL, p-tau > 60 pg/mL and Aß42 < 550 pg/mL); (ii) 121 participants in the Wisconsin Registry for Alzheimer's Prevention or Wisconsin Alzheimer's Disease Research study (84 participants cognitively unimpaired (CU) enriched for a parental history of AD, 20 participants with mild cognitive impairment (MCI), and 17 with AD); (iii) a neurologically normal cohort aged 23-78 years with paired CSF and serum samples. CSF ANGPT-2, sPDGFRß, albumin and fibrinogen levels were measured by sandwich ELISA. In cohort (i), CSF ANGPT-2 was elevated in AD and correlated with CSF t-tau and p-tau181 but not Aß42. ANGPT-2 also correlated positively with CSF sPDGFRß and fibrinogen - markers of pericyte injury and BBB leakiness. In cohort (ii), CSF ANGPT-2 was highest in MCI and correlated with CSF albumin in the CU and MCI cohorts but not in AD. CSF ANGPT-2 also correlated with CSF t-tau and p-tau and with markers of neuronal injury (neurogranin and α-synuclein) and neuroinflammation (GFAP and YKL-40). In cohort (iii), CSF ANGPT-2 correlated strongly with the CSF/serum albumin ratio. Serum ANGPT-2 showed non-significant positive associations with CSF ANGPT-2 and the CSF/serum albumin ratio. Together, these data indicate that CSF and possibly serum ANGPT-2 is associated with BBB leakiness in early AD and is closely related to tau pathology and neuronal injury. The utility of serum ANGPT-2 as a biomarker of BBB damage in AD requires further study.

A new look at angiogenesis inhibition of geniposide in experimental arthritis by blocking angiopoietin-2 exocytosis.

Angiogenesis is a key player in the pathogenesis of rheumatoid arthritis. Exocytosis from Weibel-Palade bodies is a prerequisite for angiopoietin-2 (Ang-2) to activate endothelial cells and initiate angiogenesis. Geniposide (GE) was previously reported to exert anti-angiogenic effects. The aim of this study was to shed light on whether and how GE regulates Ang-2 exocytosis. A rat model of adjuvant arthritis (AA) was established to evaluate the therapeutic effect of GE (60 and 120 mg/kg) especially in synovial angiogenesis. In addition, the Matrigel plug assay was used to detect the effect of GE (120 and 240 mg/kg) on angiogenesis in AA mice. In vitro, sphingosine-1-phosphate (S1P)-stimulated human umbilical vein endothelial cells (HUVECs) were used to investigate the effect and mechanism of GE on Ang-2 exocytosis. It was found that GE improved the symptoms of AA rats and inhibited angiogenesis in AA, which may be related to the down-regulation of S1P receptors 1, 3 (S1PR1, S1PR3), phospholipase Cß3 (PLCß3), inositol 1,4,5-trisphosphate receptor (IP3 R) and Ang-2 expression. The results of in vitro experiments showed that S1P induced rapid release of Ang-2 from HUVECs with multigranular exocytosis. Suppression of the S1P/S1PR1/3/PLCß3/Ca2+ signal axis by the S1PR1/3 inhibitor VPC23019 and the IP3 R inhibitor 2-APB blocked Ang-2 exocytosis, accompanied by diminished angiogenesis in vitro. GE dose-dependently weakened S1P/S1PR1/3/PLCß3/Ca2+ signal axis activation, Ang-2 exocytosis and angiogenesis in HUVECs (p < 0.05, p < 0.01). Overall, these findings revealed that angiogenesis inhibition of GE was partly attributed to the intervention of Ang-2 exocytosis through negatively modulating the S1P/S1PR1/3/PLCß3/Ca2+ signal axis, providing a novel strategy for rheumatoid arthritis anti-angiogenic therapy.

Endothelial Biomarkers in Critically Ill COVID-19 Patients: Potential Predictors of the Need for Dialysis.

INTRODUCTION: The aim of this was to evaluate the function of vascular biomarkers to predict the need for hemodialysis in critically ill patients with COVID-19. METHODS: This is a prospective study with 58 critically ill patients due to COVID-19 infection. Laboratory tests in general and vascular biomarkers, such as VCAM-1, syndecan-1, angiopoietin-1, and angiopoietin-2, were quantified on intensive care unit (ICU) admission. RESULTS: There was a 40% death rate. VCAM and Ang-2/Ang-1 ratio on ICU admission were associated with the need for hemodialysis. Vascular biomarkers (VCAM-1, syndecan-1, angiopoietin-2/angiopoietin-1 ratio) were predictors of death and their cutoff values were useful to stratify patients with a worse prognosis. In the multivariate cox regression analysis with adjusted models, VCAM-1 (OR 1.13 [CI 95%: 1.01-1.27]; p = 0.034) and Ang-2/Ang-1 ratio (OR 4.87 [CI 95%: 1.732-13.719]; p = 0.003) were associated with the need for dialysis. CONCLUSION: Vascular biomarkers, mostly VCAM-1 and Ang-2/Ang-1 ratio, showed better efficiency to predict the need for hemodialysis in critically ill COVID-19 patients.

Associations of Angiopoietin 2 and Vascular Endothelial Growth Factor-A Concentrations with Clinical End Points.

BACKGROUND: Angiopoietin 2 regulates endothelial function partially mediated by vascular endothelial growth factor-A (VEGF-A) and may play a role in diabetic kidney disease (DKD). We assessed the association of angiopoietin 2 and VEGF-A with cardiorenal outcomes and investigated the effect of canagliflozin on angiopoietin 2 and VEGF-A concentrations. METHODS: Two thousand five hundred sixty-five study participants with DKD and available plasma samples treated with canagliflozin or placebo in the Canagliflozin and Kidney Events in Diabetes with Established Nephropathy Clinical Evaluation (CREDENCE) trial were included. Angiopoietin 2 and VEGF-A concentrations were measured at baseline, year 1, and year 3. The primary composite end point of the trial was a composite of kidney failure, doubling of the serum creatinine level, and kidney or cardiovascular death. RESULTS: Patients with the highest baseline quartile of angiopoietin 2, but not VEGF-A, concentration had the highest risk clinical profile. Treatment with canagliflozin significantly lowered concentrations of angiopoietin 2 (adjusted geometric mean ratio: 0.94; 95% confidence interval, 0.92 to 0.95; P < 0.001), but not VEGF-A. In multivariable-adjusted modeling, each 50% increment in log baseline angiopoietin 2 concentrations was associated with a higher risk of primary composite outcome (hazard ratio, 1.27; 95% confidence interval, 1.13 to 1.43). Angiopoietin 2 change at year 1 compared with baseline explained 10% of the effect of canagliflozin on the primary composite outcome. VEGF-A concentrations were not associated with outcomes, alone or in combination with angiopoietin 2. CONCLUSIONS: Higher angiopoietin 2 levels were associated with cardiorenal risk among individuals with DKD independent of VEGF-A. Canagliflozin lowered angiopoietin 2 concentrations. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Evaluation of the Effects of Canagliflozin on Renal and Cardiovascular Outcomes in Participants With Diabetic Nephropathy, NCT02065791 .

5α-Hydroxycostic acid inhibits choroidal neovascularization in rats through a dual signalling pathway mediated by VEGF and angiopoietin 2.

BACKGROUND: 5α-Hydroxycostic acid is a eudemane sesquiterpene that is isolated from the natural plant, Laggera alata. It exerts anti-inflammatory and anti-angiogenic effects on human breast cancer cells, but its role and underlying mechanism in choroidal neovascularization (CNV) are still unclear. We conducted a study to verify that 5α-Hydroxycostic acid can inhibit the formation and leakage of CNV, and describe the possible dual pathway by which it exerts its inhibitory effects in this process. METHODS: An in vitro model of choroidal neovascularization was established using VEGF164, while a rat model of choroidal neovascularization was established using a 532 nm laser. In both models, the effects of 5α-Hydroxycostic acid in vivo and in vitro were evaluated to determine its inhibitory effect on abnormal cell proliferation, migration and tubule formation, as well as its effect on pathological changes in choroidal tissues and the area of neovascularization leakage in rats. The levels of components in the VEGF/VEGFR and Ang2/Tie2 signaling pathways were measured in tissues and cells. RESULTS: In vitro experiments have shown that 5α-Hydroxycostic acid can inhibit abnormal cell proliferation, migration and angiogenesis. Additionally, 5α-Hydroxycostic acid enhances cell adhesion by inhibiting the phosphorylation pathways of VEGFR2 and Tie2. In vivo experiments demonstrated that 5α-Hydroxycostic acid has a positive therapeutic effect on choroidal neovascularization in rats. It can effectively reduce vascular leakage, consistent with the results of the cell experiments. CONCLUSION: 5α-Hydroxycostic acid can inhibit choroidal neovascularization by interfering with the VEGF- and Ang2/Tie2-related pathways, and it may be a good candidate drug for treating CNV.

The possible dual role of Ang-2 in the prognosis of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) features a dense desmoplastic stroma, which raises the intratumoral interstitial pressure leading to vascular collapse and hypoxia, inducing angiogenesis. Vascular growth factors, such as vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2), increase in PDAC. A high VEGF and a high circulating Ang-2 associate with shorter survival in PDAC. In addition to the circulatory Ang-2, PDAC endothelial and epithelial cells express Ang-2. No correlation between tumor epithelial nor endothelial cell Ang-2 expression and survival has been published. We aimed to examine Ang-2 expression and survival. This study comprised PDAC surgical patients at Helsinki University Hospital in 2000-2013. Ang-2 immunohistochemistry staining was completed on 168 PDAC patient samples. Circulating Ang-2 levels were measured using ELISA in the sera of 196 patients. Ang-2 levels were assessed against clinical data and patient outcomes. A low tumor epithelial Ang-2 expression predicted shorter disease-specific survival (DSS) compared with a high expression (p = 0.003). A high serum Ang-2 associated with shorter DSS compared with a low circulating Ang-2 (p = 0.016). Ang-2 seemingly plays a dual role in PDAC survival. Further studies are needed to determine the mechanisms causing tumor cell Ang-2 expression and its positive association with survival.

Female sex protects against renal edema, but not lung edema, in mice with partial deletion of the endothelial barrier regulator Tie2 compared to male sex.

BACKGROUND: The endothelial angiopoietin/Tie2 system is an important regulator of endothelial permeability and targeting Tie2 reduces hemorrhagic shock-induced organ edema in males. However, sexual dimorphism of the endothelium has not been taken into account. This study investigated whether there are sex-related differences in the endothelial angiopoietin/Tie2 system and edema formation. METHODS: Adult male and female heterozygous Tie2 knockout mice (Tie2+/-) and wild-type controls (Tie2+/+) were included (n = 9 per group). Renal and pulmonary injury were determined by wet/dry weight ratio and H&E staining of tissue sections. Protein levels were studied in plasma by ELISA and pulmonary and renal mRNA expression levels by RT-qPCR. RESULTS: In Tie2+/+ mice, females had higher circulating angiopoietin-2 (138%, p<0.05) compared to males. Gene expression of angiopoietin-1 (204%, p<0.01), angiopoietin-2 (542%, p<0.001) were higher in females compared to males in kidneys, but not in lungs. Gene expression of Tie2, Tie1 and VE-PTP were similar between males and females in both organs. Renal and pulmonary wet/dry weight ratio did not differ between Tie2+/+ females and males. Tie2+/+ females had lower circulating NGAL (41%, p<0.01) compared to males, whereas renal NGAL and KIM1 gene expression was unaffected. Interestingly, male Tie2+/- mice had 28% higher renal wet/dry weight ratio (p<0.05) compared to Tie2+/+ males, which was not observed in females nor in lungs. Partial deletion of Tie2 did not affect circulating angiopoietin-1 or angiopoietin-2, but soluble Tie2 was 44% and 53% lower in males and females, respectively, compared to Tie2+/+ mice of the same sex. Renal and pulmonary gene expression of angiopoietin-1, angiopoietin-2, estrogen receptors and other endothelial barrier regulators was comparable between Tie2+/- and Tie2+/+ mice in both sexes. CONCLUSION: Female sex seems to protect against renal, but not pulmonary edema in heterozygous Tie2 knock-out mice. This could not be explained by sex dimorphism in the endothelial angiopoietin/Tie2 system.

Relationship of angiopoietin-2 level with the prognosis of the COVID-19 disease.

INTRODUCTION: COVID-19 infection is associated with coagulopathy. There is increased expression of markers such as E-selectin or angiopoietin-2 upon the activation of endothelin. The aim of this study was to determine whether there was a difference in angiopoietin-2 levels among patients with a diagnosis of COVID-19 who need to be hospitalized in the intensive care units (ICUs) or service. METHODOLOGY: COVID-19 infected patients admitted in the hospital were included in this study. In addition to the routine biochemical parameters of patients in ICUs and services, 5 cc blood samples were collected and angiopoietin-2 was analyzed. Demographic data of the patients, biochemical parameters at the time of hospitalization, places and durations of hospitalization as well as their ways of being discharged from hospital were recorded. RESULTS: 180 patients who presented to our hospital's emergency service and were hospitalized with a diagnosis of COVID-19 were included in our study. 137 patients (76.1%) were hospitalized in the service and 43 (23.9%) were hospitalized in ICU. The angiopoietin-2 level was determined to be significantly high in the patients hospitalized in ICUs (p = 0.018). When the cut-off value of angiopoetin-2 in predicting the ICU hospitalization was assumed as 64.5 ng/L, its sensitivity was determined to be 59% and its specificity was found to be 42%. CONCLUSIONS: We concluded that angiopoietin-2 level in COVID-19 patients upon their presentation to the hospital might be an important parameter in predicting and ascertaining their place of hospitalization.

Therapeutic targeting of angiopoietins in tumor angiogenesis and cancer development.

The formation and progression of tumors in humans are linked to the abnormal development of new blood vessels known as neo-angiogenesis. Angiogenesis is a broad word that encompasses endothelial cell migration, proliferation, tube formation, and intussusception, as well as peri-EC recruitment and extracellular matrix formation. Tumor angiogenesis is regulated by angiogenic factors, out of which some of the most potent angiogenic factors such as vascular endothelial growth factor and Angiopoietins (ANGs) in the body are produced by macrophages and other immune cells within the tumor microenvironment. ANGs have a distinct function in tumor angiogenesis and behavior. ANG1, ANG 2, ANG 3, and ANG 4 are the family members of ANG out of which ANG2 has been extensively investigated owing to its unique role in modifying angiogenesis and its tight association with tumor progression, growth, and invasion/metastasis, which makes it an excellent candidate for therapeutic intervention in human malignancies. ANG modulators have demonstrated encouraging outcomes in the treatment of tumor development, either alone or in conjunction with VEGF inhibitors. Future development of more ANG modulators targeting other ANGs is needed. The implication of ANG1, ANG3, and ANG4 as probable therapeutic targets for anti-angiogenesis treatment in tumor development should be also evaluated. The article has described the role of ANG in tumor angiogenesis as well as tumor growth and the treatment strategies modulating ANGs in tumor angiogenesis as demonstrated in clinical studies. The pharmacological modulation of ANGs and ANG-regulated pathways that are responsible for tumor angiogenesis and cancer development should be evaluated for the development of future molecular therapies.

Involvement of Angiopoietin 2 and vascular endothelial growth factor in uveitis.

PURPOSE: Angiopoietin (Ang) 2 is released from vascular endothelial cells by the stimulation of vascular endothelial growth factor (VEGF)A. Ang2 increases the expression of leukocyte adhesion molecules on endothelial cells via nuclear factor κB. The aim of this study was to evaluate the effects of Ang2 and VEGFA on ocular autoimmune inflammation. METHODS: We measured the concentrations of Ang2 and VEGFA in vitreous samples among patients with uveitis. Vitreous samples were collected from 16 patients with idiopathic uveitis (uveitis group) and 16 patients with non-inflammatory eye disease (control group). Experimental autoimmune uveoretinitis (EAU) was induced in B10.BR mice with a human interphotoreceptor retinoid-binding protein-derived peptide. The retinochoroidal tissues of the EAU mice were removed, and the mRNA levels of Ang2 and VEGFA were examined. EAU mice treated with anti-Ang2, anti-VEGFA, a combination of anti-Ang2 and anti-VEGFA, anti-Ang2/VEGFA bispecific, or IgG control antibodies were clinically and histopathologically evaluated. RESULTS: The protein levels of Ang2 and VEGFA were significantly higher in the vitreous samples of patients with uveitis than in controls (P<0.05). The retinochoroidal mRNA levels of Ang2 and VEGFA were significantly upregulated in EAU mice compared to controls (n = 6, P<0.05). Although there was no significant difference, treatment with anti-VEGFA antibody reduced the clinical and histopathological scores. However, treatment with anti-Ang2 antibody reduced the clinical and histopathological scores (n = 18-20, P<0.05). Furthermore, these scores were further decreased when treated by inhibiting both Ang2 and VEGFA. CONCLUSIONS: Based on these results, VEGFA and Ang2 were shown to be upregulated locally in the eye of both uveitis patients and models of uveitis. Dual inhibition of Ang2 and VEGFA is suggested to be a new therapeutic strategy for uveitis.

Serum Angiopoietin-2 level increase differs between polytraumatized patients with and without central nervous system injuries.

Since endothelial cells rapidly release Angiopoietin-2 (Ang-2) in response to vascular injury and inflammatory stimuli, we aimed to investigate if its serum levels increase in polytraumatized patients. Our cohort study evaluated 28 blunt polytrauma survivors (mean age, 38.4 years; median ISS, 34) who were directly admitted to our level I trauma center in 2018. We assessed the serum Ang-2 level at admission and on days 1, 3, 5, 7, and 10 during hospitalization. Ang-2 was released into the circulation immediately after polytrauma. At admission (day 0), it amounted to 8286 ± 5068 pg/mL, three-and-a-half times the reference value of 2337 ± 650 pg/mL assessed in a healthy control group. Subgroup analysis provided a higher mean Ang-2 level in the CNSI group combining all patients suffering a brain or spinal cord injury compared to the non-CNSI group solely on day 0 [11083 ± 5408 pg/mL versus 3963 ± 2062 pg/mL; p < 0.001]. Whereas the mean Ang-2 level increased only in the non-CNSI group from day 0 to day 3 (p = 0.009), the respective curves showed similar continuous decreases starting with day 3. Multivariate logistic regression analysis revealed an association between the Ang-2 day 0 level and the presence of a CNSI (OR = 1.885; p = 0.048). ROC analysis provided a cutoff level of 5352 pg/mL. In our study group, serum Ang-2 levels assessed at admission differed between polytraumatized patients with and without brain or spinal cord injuries. Based on our findings, we consider serum Ang-2 levels an effective biomarker candidate for indicating CNSI in these patients at admission, worthy of further evaluation in large multicenter studies.

The impact of early RPE cell junction loss on VEGF, Ang-2, and TIMP secretion in vitro.

Purpose: The retinal pigment epithelium (RPE) is an important tissue for maintaining a healthy retina. Retinal pigment epithelial cells help regulate nutrient transport to photoreceptors and are heavily pigmented to prevent light scattering. These cells also have junction proteins to form monolayers. Monolayers are key players in pathologies such as age-related macular degeneration (AMD), a leading cause of vision loss in older adults. During AMD, RPE cell detachment can occur, resulting in a loss of junctions. Losing junctions can increase the expression of pro-angiogenic vascular endothelial growth factor (VEGF). This overexpression can cause abnormal blood vessel growth or angiogenesis in the retina. Age-related macular degeneration treatments target VEGF to slow angiogenesis progression. However, other proteins, such as angiopoietin-2 (Ang-2) and the tissue inhibitor of metalloproteinase-1 (TIMP-1), may also play important roles, making them potential targets for treatment. Controlling RPE junction formation will help elucidate the relationship between RPE cell detachment and additional angiogenic factor secretion, lead to more therapeutics, and increase the efficacy of current treatments. Methods: Micropatterning was used to control the spatial arrangement of primary porcine RPE cells using polydimethylsiloxane (PDMS) stencils. Patterns were formed into PDMS stencils to mimic 10%, 25%, and 50% overall detachment of the RPE monolayer. Zonula-occludens-1 (ZO-1), Ang-2, and VEGF were visualized using immunocytochemical (ICC) staining. An enzyme-linked immunosorbent assay (ELISA) was used to quantify extracellular Ang-2, VEGF, TIMP-1, and TIMP-2 levels. A rod outer segment (OS) phagocytosis assay was performed to determine how RPE junction loss directly affects photoreceptor support. Results: The growth of primary porcine RPE cells was successfully controlled using stencils. Morphological changes and a decrease in pigmentation were observed, showing a decline in barrier and light absorption functions as degeneration increased. One day after stencil removal, junction proteins were delocalized, and angiogenic factor secretions were correlated with increased levels of detachment. Secretion levels of Ang-2 and TIMP-1 were significantly increased, whereas VEGF and TIMP-2 concentrations were not as affected by varying levels of detachment. OS phagocytosis appeared lower in RPE cells when ZO-1 was affected. Conclusions: These results suggest a correlation between loss of junctions, abnormal angiogenic protein secretion, and reduced OS phagocytosis. Furthermore, Ang-2 and TIMP-1 proteins might be beneficial targets for AMD treatments, and their roles in retinal diseases deserve further investigation.

Expression of Angiopoietin-2 in Lung Tissue of Juvenile SD Rats with Lipopolysaccharide-Induced Acute Lung Injury and the Role of Ulinastatin.

This study aimed to observe the expression of angiopoietin-2 (Ang-2) in the lung tissue of juvenile SD rats with lipopolysaccharide (LPS)-induced acute lung injury (ALI) and to clarify the role of ulinastatin (UTI). Ninety 18-21-day-old juvenile SD male rats were randomly divided into five groups (n = 18). ALI rat model was established by intraperitoneal injection of LPS (LPS 10 mg/kg), while the control group was given the same dose of normal saline. The UTI intervention group was given the injection of UTI (5000 U/mL) immediately after the injection of LPS, which was divided into UTI low-dose group (LPS + 5 ml/kg UTI), UTI medium-dose group (LPS + 10 ml/kg UTI), and UTI high-dose group (LPS + 20 ml/kg UTI).The respiratory status of each group of rats was observed, and six rats were randomly selected to be killed in each group at 6, 12, and 24 h, and the lung tissues were dissected and retained. The pathological changes of the lung tissues were observed by hematoxylin-eosin (HE) staining, the expression levels and locations of Ang-2 and vascular endothelial growth factor (VEGF) in lung tissue were observed by immunohistochemical staining, and the expressions of genes and proteins of Ang-2 and VEGF were detected by quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. Three hours after intraperitoneal injection, rats in the model group developed shortness of breath and the developed respiratory distress progressed over time. The lung pathological changes in the model group were obvious compared with those in the control group, and gradually worsened with time, and the pathological changes of lung in the rats in the UTI intervention group were reduced compared with those in the model group. At different time points, the expressions of Ang-2 and VEGF in the lung tissue of rats in the model group were higher than those in the control group, and were lower in the UTI intervention group than those in the model group. The expressions of Ang-2 and VEGF protein were lower in the low-dose group of UTI group than those in the high-dose group of UTI group at different time points (P < 0.05), and the expressions of Ang-2 and VEGF protein in the low-dose group of UTI were significantly lower than those in the medium-dose group at 12 h and 24 h (P < 0.05). The expression of Ang-2 was increased in the lung tissue of juvenile SD rats with LPS-induced ALI, and was associated with the degree of lung injury. UTI might attenuate LPS-induced ALI by inhibiting the expression of Ang-2 in lung tissue, and the low dose was more obvious than the medium and high dose.

An angiopoietin 2, FGF23, and BMP10 biomarker signature differentiates atrial fibrillation from other concomitant cardiovascular conditions.

Early detection of atrial fibrillation (AF) enables initiation of anticoagulation and early rhythm control therapy to reduce stroke, cardiovascular death, and heart failure. In a cross-sectional, observational study, we aimed to identify a combination of circulating biomolecules reflecting different biological processes to detect prevalent AF in patients with cardiovascular conditions presenting to hospital. Twelve biomarkers identified by reviewing literature and patents were quantified on a high-precision, high-throughput platform in 1485 consecutive patients with cardiovascular conditions (median age 69 years [Q1, Q3 60, 78]; 60% male). Patients had either known AF (45%) or AF ruled out by 7-day ECG-monitoring. Logistic regression with backward elimination and a neural network approach considering 7 key clinical characteristics and 12 biomarker concentrations were applied to a randomly sampled discovery cohort (n = 933) and validated in the remaining patients (n = 552). In addition to age, sex, and body mass index (BMI), BMP10, ANGPT2, and FGF23 identified patients with prevalent AF (AUC 0.743 [95% CI 0.712, 0.775]). These circulating biomolecules represent distinct pathways associated with atrial cardiomyopathy and AF. Neural networks identified the same variables as the regression-based approach. The validation using regression yielded an AUC of 0.719 (95% CI 0.677, 0.762), corroborated using deep neural networks (AUC 0.784 [95% CI 0.745, 0.822]). Age, sex, BMI and three circulating biomolecules (BMP10, ANGPT2, FGF23) are associated with prevalent AF in unselected patients presenting to hospital. Findings should be externally validated. Results suggest that age and different disease processes approximated by these three biomolecules contribute to AF in patients. Our findings have the potential to improve screening programs for AF after external validation.

Angiopoietin-2 blockade suppresses growth of liver metastases from pancreatic neuroendocrine tumors by promoting T cell recruitment.

Improving the management of metastasis in pancreatic neuroendocrine tumors (PanNETs) is critical, as nearly half of patients with PanNETs present with liver metastases, and this accounts for the majority of patient mortality. We identified angiopoietin-2 (ANGPT2) as one of the most upregulated angiogenic factors in RNA-Seq data from human PanNET liver metastases and found that higher ANGPT2 expression correlated with poor survival rates. Immunohistochemical staining revealed that ANGPT2 was localized to the endothelial cells of blood vessels in PanNET liver metastases. We observed an association between the upregulation of endothelial ANGPT2 and liver metastatic progression in both patients and transgenic mouse models of PanNETs. In human and mouse PanNET liver metastases, ANGPT2 upregulation coincided with poor T cell infiltration, indicative of an immunosuppressive tumor microenvironment. Notably, both pharmacologic inhibition and genetic deletion of ANGPT2 in PanNET mouse models slowed the growth of PanNET liver metastases. Furthermore, pharmacologic inhibition of ANGPT2 promoted T cell infiltration and activation in liver metastases, improving the survival of mice with metastatic PanNETs. These changes were accompanied by reduced plasma leakage and improved vascular integrity in metastases. Together, these findings suggest that ANGPT2 blockade may be an effective strategy for promoting T cell infiltration and immunostimulatory reprogramming to reduce the growth of liver metastases in PanNETs.