Tie2 Research Articles

MiR-1233-3p Inhibits Angiopoietin-1-Induced Endothelial Cell Survival, Migration, and Differentiation.

Angiopoietin-1 (Ang-1) and its receptor Tie-2 promote vascular integrity and angiogenesis. MicroRNAs (miRNAs) are involved in the regulation of many cellular functions, including endothelial cell (EC) survival, proliferation, and differentiation. Several reports indicate that these effects of miRNAs on EC functions are mediated through the modulation of angiogenesis factor signaling including that of vascular endothelial growth factor (VEGF). To date, very little is known about the roles played by miRNAs in the signaling and angiogenesis promoted by the Ang-1-Tie-2 receptor axis. Our high-throughput screening of miRNAs regulated by Ang-1 exposure in human umbilical vein endothelial cells (HUVECs) has identified miR-1233-3p as a mature miRNA whose cellular levels are significantly downregulated in response to Ang-1 exposure. The expression of miR-1233-3p in these cells is also downregulated by other angiogenesis factors including VEGF, fibroblast growth factor 2 (FGF-2), transforming growth factor β (TGFβ), and angiopoietin-2 (Ang-2). The overexpression of miR-1233-3p in HUVECs using specific mimics significantly attenuated cell survival, migration, and capillary-like tube formation, and promoted apoptosis. Moreover, miR-1233-3p overexpression resulted in reversal of the anti-apoptotic, pro-migration, and pro-differentiation effects of Ang-1. Biotinylated miRNA pull-down assays showed that p53 and DNA damage-regulated 1 (PDRG1) is a direct target of miR-1233-3p in HUVECs. The exposure of HUVECs to Ang-1, angiopoietin-2 (Ang-2), fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF), or transforming growth factor β (TGFβ) triggers the regulation of PDRG1 expression. This study highlights that miR-1233-3p exerts inhibitory effects on Ang-1-induced survival, migration, and the differentiation of cultured ECs.

Cardiovascular Risk Biomarkers in Women with and Without Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is a prevalent metabolic disorder with an increased risk for cardiovascular disease (CVD) that is enhanced by obesity. This study sought to determine whether a panel of cardiovascular risk proteins (CVRPs) would be dysregulated in overweight/obese PCOS patients, highlighting potential biomarkers for CVD in PCOS. In this exploratory cross-sectional study, plasma levels of 54 CVRPs were analyzed in women with PCOS (n = 147) and controls (n = 97). CVRPs were measured using the SOMAscan proteomic platform (version 3.1), with significant proteins identified through linear models, regression analysis, and receiver operating characteristic (ROC) analysis. Analysis on BMI-matched subsets of the cohort were undertaken. Functional enrichment and protein-protein interaction analyses elucidated the pathways involved. Eleven CVRPs were dysregulated in PCOS (whole set, without matching for body mass index (BMI) or insulin resistance (IR)): leptin, Interleukin-1 receptor antagonist protein (IL-1Ra), polymeric immunoglobulin receptor (PIGR), interleukin-18 receptor (IL-18Ra), C-C motif chemokine 3 (MIP-1a), and angiopoietin-1 (ANGPT1) were upregulated whilst advanced glycosylation end product-specific receptor, soluble (sRAGE), bone morphogenetic protein 6 (BMP6); growth/differentiation factor 2 (GDF2), superoxide dismutase [Mn] mitochondrial (MnSOD), and SLAM family member 5 (SLAF5) were downregulated versus the controls. In BMI-matched (overweight/obese, BMI ≥ 26 kg/m2) subset analysis, six CVRPs were common to the whole set: ANGPT1 and IL-1Ra were upregulated; and sRAGE, BMP6, GDF2, and Mn-SOD were downregulated. In addition, lymphotactin (XCL1) was upregulated and placenta growth factor (PIGF), alpha-L-iduronidase (IDUA), angiopoietin-1 receptor, and soluble (sTie-2) and macrophage metalloelastase (MMP12) were downregulated. A subset analysis of BMI-matched plus insulin resistance (IR)-matched women revealed only upregulation of tissue factor (TF) and renin in PCOS, potentially serving as biomarkers for cardiovascular risk in overweight/obese women with PCOS. A combination of upregulated obesity-related CVRPs (ANGPT1/IL/1Ra/XCL1) and downregulated cardioprotective proteins (sRAGE/BMP6/Mn-SOD/GDF2) in overweight/obese PCOS women may contribute to the increased risk for CVD. TF and renin upregulation observed in the BMI- and IR-matched limited sample PCOS subgroup indicates their potential risk of CVD.

RPL36-mediated selective loading of miR-4432 into extracellular vesicles contributes to perivascular cell dysfunction in venous malformations.

Venous malformations (VMs), predominantly arising from activating mutations of tyrosine kinase receptor TIE2 within endothelial cells (ECs), are characterized by dilated and tortuous vessels with a paucity of perivascular cells. The mechanisms of interaction between mutant ECs and perivascular cells remain largely elusive. To investigate the characteristics of extracellular vesicles (EVs) from VM ECs, especially their carried miRNAs and their roles in the crosstalk between ECs and perivascular cells in VM pathogenesis. MiRNA profiles of human umbilical vein endothelial cells overexpressing TIE2L914F (L914F cells) and TIE2WT (WT cells) along with their EVs were analyzed using RNA sequencing. In vitro studies using umbilical cord stem cells (UCSCs) were performed for the functional assays of VM EVs and their enriched microRNA-4432 (miR-4432). MiRNA-pulldown and RNA interference techniques were employed to identify the sorting regulator of miR-4432 into VM EVs. We observed an upregulation of RNA secretion in L914F EVs compared to WT EVs. MiRNA sequencing revealed a distinguished profile of L914F EVs compared to L914F cells, WT cells, and WT EVs, identifying miR-4432 as preferentially encapsulated within EVs from L914F cells. Functional assays demonstrated that VM EVs and EV-carried miR-4432 inhibited the differentiation, adhesion, and proliferation of UCSCs. Furthermore, RPL36 was identified as an RNA binding protein and sorting regulator of miR-4432 during the EV secretion process in L914F cells. This study, for the first time, identifies the interaction between VM ECs and perivascular cells via EVs, and offers valuable data on the miRNA profiles of VM ECs and normal ECs, along with their EVs. Our findings suggest that the RPL36-mediated selective loading of miR-4432 into EVs may contribute to the aberrant perivascular cell coverage in VMs, providing novel insights into VM pathogenesis and potential treatment strategies.

Angiogenic effects of Type 2 diabetes on the dental pulp.

To investigate the influence of type 2 diabetes (T2D) and hyperglycaemia on blood vessels and angiogenic markers in the dental pulp. Extracted non-carious permanent molar teeth were collected from patients with well-controlled T2D (n = 10) and non-T2D (controls) (n = 10). The pulp was examined qualitatively using haematoxylin and eosin and Van Gieson stains. Immunohistochemistry (IHC) identified the primary receptor for VEGF, VEGFR2, and the endothelial cell marker CD34. Primary human dental pulp cell (hDPC) lines (n = 3) were established from tissue explants and cells were grown in media containing 5.5 mM D-glucose (control), 12.5 mM (prediabetes) and 25 mM (T2D) D-glucose under normoxic conditions for 24, 48 and 72 h. Assays for metabolic activity (PrestoBlue) and cell viability (Crystal Violet staining) assessed the hDPC response to hyperglycaemia. The expression of angiogenic genes VEGFA, KDR, FLT-1, ANGPT1, ANGPT2, TIE1 and TEK were analysed using quantitative real-time polymerase chain reaction. ELISAs were used to quantify the level of expressed protein for VEGFA, ANG1, ANG2, TIE1, and TIE2 in the media. Data analyses were performed using GraphPad Prism and anova tests at p < .05. Blood vessels in T2D samples had thicker walls and stained strongly for elastin and collagen compared with non-T2D samples. VEGFR2 protein was not seen in any T2D samples but consistently detected in healthy specimens. Culturing healthy cells in high glucose (25 mM) significantly reduced cell viability at 24 h compared to the control (p = .005) and 12.5 mM glucose (p = .001) but the metabolic activity was not greatly affected by glucose and time. VEGFA mRNA and VEGFA protein expression were detected in the hDPCs in the presence of hyperglycaemia over time; however, the primary receptor, VEGFR2/KDR, was not detected. Genes for the ANG1 and ANG2 and their receptors were expressed at all glucose concentrations but hyperglycaemia upregulated ANG2 mRNA. Proteins for all growth factors were detected in the media however proteins for TIE1 and TIE2 receptors were not. T2D and hyperglycaemia may impair the angiogenic response in the pulp similar to other body site. The scarcity of VEGFR2 and increased expression of ANG2 in response to hyperglycaemia suggests that VEGF and ANG-Tie1/Tie2 signalling may be compromised.

Endothelial FOSL1 drives angiotensin II-induced myocardial injury via AT1R-upregulated MYH9.

Vascular remodeling represents a pathological basis for myocardial pathologies, including myocardial hypertrophy and myocardial infarction, which can ultimately lead to heart failure. The molecular mechanism of angiotensin II (Ang II)-induced vascular remodeling following myocardial infarction reperfusion is complex and not yet fully understood. In this study, we examined the effect of Ang II infusion on cardiac vascular remodeling in mice. Single-cell sequencing showed Ang IIinduced cytoskeletal pathway enrichment and that FOS like-1 (FOSL1) affected mouse cardiac endothelial dysfunction by pseudotime analysis. Myosin heavy chain 9 (MYH9) was predominantly expressed in primary cardiac endothelial cells. The Ang II type I receptor blocker telmisartan and the protein kinase C inhibitor staurosporine suppressed Ang II-induced upregulation of MYH9 and FOSL1 phosphorylation in human umbilical vein endothelial cells. Silencing MYH9 abolished Ang II-mediated inhibition of angiogenesis in human umbilical vein endothelial cells, and attenuated AngII-induced vascular hyperpermeability. We found that FOSL1 directly bound to the MYH9 promoter and thus activated transcription of MYH9 by the dual luciferase reporter and chromatin immunoprecipitation assays, leading to vascular dysfunction. In vivo, 6 weeks after injecting adeno-associated virus-ENT carrying the TEK tyrosine kinase (tie) promoter-driven short hairpin RNA for silencing FOSL1 (AAV-tie-shFOSL1), cardiac function represented by the ejection fraction and fractional shortening was improved, myocardial fibrosis was decreased, protein levels of phosphorylated FOSL1, MYH9, and collagen type I alpha were reduced, and cardiac vascular density was recovered in mice with endothelial Fosl1-specific knockdown in Ang II-infused mice. In ischemia-reperfusion mice, AAV-shFosl1 mice had a reduced infarct size and preserved cardiac function compared with control AAV mice. Our findings suggest a critical role of the FOSL1/MYH9 axis in hindering Ang II-induced vascular remodeling, and we identified FOSL1 as a potential therapeutic target in endothelial cell injuries induced by myocardial ischemia-reperfusion.

CNSC-30. DETERMINING THE REGULATION AND FUNCTION OF ANGPT1 HIGH-GRADE GLIOMAS

Abstract Angiopoietin-1 (Angpt1) is a secreted protein that promotes angiogenesis and vascular stability in development and certain disease states through activation of the Tie2 receptor. However, little is known about its regulation and function in brain tumors. Angpt1 is upregulated in glial brain tumors, including high-grade gliomas and ependymomas. We find Angpt1 is expressed by specific cell populations within gliomas, along with non-tumor reactive astrocytes and perivascular mural cells. Leveraging an in vivo brain tumor modeling system we have generated glioma models to investigate how tumor cell state impacts Angpt1 expression, along with creating Angpt1 knock-out models. We find that Angpt1 expression is increased when glioma models are biased towards a more astrocytic and/or mesenchymal state, mirroring developmental expression patterns. Ongoing studies utilizing these models are being used to determine the mechanisms that regulate the dynamic expression of Angpt1 and its function in glioma pathogenesis, including tumor angiogenesis and blood-brain barrier function.

Molecular and Cellular Mechanisms Involved in the Pathophysiology of Retinal Vascular Disease-Interplay Between Inflammation and Oxidative Stress.

Retinal vascular diseases encompass several retinal disorders, including diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, and retinal vascular occlusion; these disorders are classified as similar groups of disorders due to impaired retinal vascularization. The aim of this review is to address the main signaling pathways involved in the pathogenesis of retinal vascular diseases and to identify crucial molecules and the importance of their interactions. Vascular endothelial growth factor (VEGF) is recognized as a crucial and central molecule in abnormal neovascularization and a key phenomenon in retinal vascular occlusion; thus, anti-VEGF therapy is now the most successful form of treatment for these disorders. Interaction between angiopoietin 2 and the Tie2 receptor results in aberrant Tie2 signaling, resulting in loss of pericytes, neovascularization, and inflammation. Notch signaling and hypoxia-inducible factors in ischemic conditions induce pathological neovascularization and disruption of the blood-retina barrier. An increase in the pro-inflammatory cytokines-TNF-α, IL-1β, and IL-6-and activation of microglia create a persistent inflammatory milieu that promotes breakage of the blood-retinal barrier and neovascularization. Toll-like receptor signaling and nuclear factor-kappa B are important factors in the dysregulation of the immune response in retinal vascular diseases. Increased production of reactive oxygen species and oxidative damage follow inflammation and together create a vicious cycle because each factor amplifies the other. Understanding the complex interplay among various signaling pathways, signaling cascades, and molecules enables the development of new and more successful therapeutic options.

Novel postzygotic RASA1 mutation in a patient with Parkes Weber syndrome: A case report and literature review.

Not only germline but also postzygotic mutations in the RASA1 or EPHB4 genes can lead to capillary malformation-arteriovenous malformation (CM-AVM) syndrome. As it is not always possible to clinically distinguish between constitutional variants and postzygotic mosaicism, a sufficiently high sequencing depth must be used in genetic diagnostics to detect both. Capillary malformation-arteriovenous malformation (CM-AVM) syndrome, with or without Parkes Weber syndrome, is a rare autosomal dominant disease caused by pathogenic RASA1 or EPHB4 variants. Up to 80% of CM-AVM cases have an affected parent. Gene panel sequencing was performed for a 4-year-old girl with multiple CMs, two capillary stains on the left leg, and associated overgrowth of the second toe. We also reviewed published cases with mosaic RASA1 and EPHB4 mutations. A mosaic RASA1 loss-of-function mutation was detected with a variant allele frequency (VAF) of 20% in the blood and oral epithelial cells of the index patient. The literature review illustrates that the severity of the clinical phenotype does not correlate with the VAF. We also identified a germline nonsense variant in the patient's TEK gene. However, inactivating TEK variants do not cause a vascular phenotype but can confer an increased risk for primary congenital glaucoma with variable expressivity. The case presented here illustrates that the choice of the sequencing depth of a diagnostic next-generation sequencing test for CM-AVM patients should always take mosaicism into account and that a good knowledge of the sequenced genes and associated disease mechanisms is necessary for adequate genetic counseling.

Circulating Angiopoietin-2 (Angpt2) levels predict diagnosis of Chronic Thromboembolic Pulmonary Hypertension (CTEPH)

Abstract Background Vascular remodeling following incomplete thrombus resolution is considered to be a key factor in the progression to chronic thromboembolic pulmonary hypertension (CTEPH) after acute pulmonary embolism (PE). Recent data demonstrated that Angpt2, which controls signaling via the Tie2 receptor, could interfere with the normal process of thrombus resolution. Our prior findings have shown that patients presenting with acute PE and having plasma levels of Angt-2 ≥5.5 ng/ml at admission are at a significantly increased risk (93 times higher) of being diagnosed with CTEPH in subsequent assessments. Purpose To externally validate the prognostic performance of Angpt2 for the development of CTEPH. Methods In a prospective cohort study of unselected consecutive all-comers with PE, survivors of the index acute event underwent 3-, 12- and 24-month follow-up with venous blood sampling for subsequent biomarker measurements. Angpt2 was measured on admission using specific enzyme linked immunosorbent assays (ELISA) detecting both the endogenous and the recombinant protein, according to the manufacturer (R&amp;D Systems). Results Overall, 319 patients with acute PE (median age: 62 [IQR, 50-73]; females: 55.2%) were included in the analysis with a median Angpt2 plasma level of 2.6 ng/ml (IQR, 1.8-3.7) (Figure 1). Patients with PE and Angpt-2 plasma levels above the median had more often comorbidities, such as chronic heart failure (24.2% and 11.0.%; p=0.045), chronic kidney disease (33.3% vs 14.0%; p=0.010), or diabetes (27.3% vs. 12.9%; p=0.032). Angpt2 plasma levels correlated with haemodynamic status and parameters indicating severe PE (right ventricle dysfunction, r=0.129 [p=0.021) and troponin, r=0.246 [p&amp;lt;0.001]). In total, post-PE impairment (PPEI) was diagnosed in 33 (10.3%) and CTEPH in four (1.3%) patients during follow-up. Angpt2 plasma levels were higher on admission in patients developing PPEI or CTEPH during follow-up compared to patients without PPEI or CTEPH (PPEI vs non-PPEI: median, 3.2 [2.3-4.1] ng/ml vs. 2.5 [1-3.4] ng/ml, p=0.035; CTEPH vs. non-CTEPH: median, 5.1 [2.9-10.5] ng/ml vs. 2.5 [1.7-3.6] ng/ml, p=0.055). Regarding the diagnosis of CTEPH during follow-up, ROC analysis illustrated an AUC for Angpt2 (0.779 [95% CI 0.56–0.99]; p=0.055]) (Figure 2). By using univariate logistic regression analysis, the calculated predefined Angpt2 cut-off value of ≥5.5 ng/ml was associated with a 9.2-fold increased risk (1.3–67.4, p=0.030) for a diagnosis of CTEPH during follow-up. Conclusion We were able to validate in a different patient cohort the usefulness of Angpt2 as a novel biomarker to identify patients at risk for CTEPH.

Angiopoietin-2 reverses endothelial cell dysfunction in progeria vasculature.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disorder in children caused by a point mutation in the lamin A gene, resulting in a toxic form of lamin A called progerin. Accelerated atherosclerosis leading to heart attack and stroke are the major causes of death in these patients. Endothelial cell (EC) dysfunction contributes to the pathogenesis of HGPS related cardiovascular diseases (CVD). Endothelial cell-cell communications are important in the development of the vasculature, and their disruptions contribute to cardiovascular pathology. However, it is unclear how progerin interferes with such communications that lead to vascular dysfunction. An antibody array screening of healthy and HGPS patient EC secretomes identified Angiopoietin-2 (Ang2) as a down-regulated signaling molecule in HGPS ECs. A similar down-regulation of Ang2 mRNA and protein was detected in the aortas from an HGPS mouse model. Addition of Ang2 to HGPS ECs rescues vasculogenesis, normalizes endothelial cell migration and gene expression, and restores nitric oxide bioavailability through eNOS activation. Furthermore, Ang2 addition reverses unfavorable paracrine effects of HGPS ECs on vascular smooth muscle cells. Lastly, by utilizing adenine base editor (ABE)-corrected HGPS ECs and progerin-expressing HUVECs, we demonstrated a negative correlation between progerin and Ang2 expression. Lastly, our results indicated that Ang2 exerts its beneficial effect in ECs through Tie2 receptor binding, activating an Akt-mediated pathway. Together, these results provide molecular insights into EC dysfunction in HGPS and suggest that Ang2 treatment has potential therapeutic effects in HGPS-related CVD.

Epigenetic regulation by polycomb repressive complex 1 promotes cerebral cavernous malformations

Cerebral cavernous malformations (CCMs) are anomalies of the cerebral vasculature. Loss of the CCM proteins CCM1/KRIT1, CCM2, or CCM3/PDCD10 trigger a MAPK-Krüppel-like factor 2 (KLF2) signaling cascade, which induces a pathophysiological pattern of gene expression. The downstream target genes that are activated by KLF2 are mostly unknown. Here we show that Chromobox Protein Homolog 7 (CBX7), component of the Polycomb Repressive Complex 1, contributes to pathophysiological KLF2 signaling during zebrafish cardiovascular development. CBX7/cbx7a mRNA is strongly upregulated in lesions of CCM patients, and in human, mouse, and zebrafish CCM-deficient endothelial cells. The silencing or pharmacological inhibition of CBX7/Cbx7a suppresses pathological CCM phenotypes in ccm2 zebrafish, CCM2-deficient HUVECs, and in a pre-clinical murine CCM3 disease model. Whole-transcriptome datasets from zebrafish cardiovascular tissues and human endothelial cells reveal a role of CBX7/Cbx7a in the activation of KLF2 target genes including TEK, ANGPT1, WNT9, and endoMT-associated genes. Our findings uncover an intricate interplay in the regulation of Klf2-dependent biomechanical signaling by CBX7 in CCM. This work also provides insights for therapeutic strategies in the pathogenesis of CCM.

Glaucoma-Protective Human Single-Nucleotide Polymorphism in the Angpt2 Locus Increased ANGPT2 Expression and Schlemm Canal Area in Mice-Brief Report.

The ANGPT (angiopoietin)-TEK (tyrosine kinase, endothelial) vascular signaling pathway plays a key role in the formation of Schlemm canal, and loss-of-function mutations in the TEK or ANGPT1 gene are associated with primary congenital glaucoma in children. In genome-wide association studies, an association was identified between protection from primary open-angle glaucoma and the single-nucleotide polymorphism rs76020419 (G>T), located within a predicted miR-145-binding site in the 3' untranslated region of ANGPT2. To date, the functional impact of this variant in the anterior chamber of the eye remains largely unexplored. MT (mutant) mice harboring an orthologous rs76020419 minor allele (T) were generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9). Plasma and tissue samples, including eyes, were collected, and ANGPT2 expression was quantified using ELISA. Anterior segments from eyes were collected from WT (wild-type) and MT mice, and Schlemm canal area was quantified. In the MT group, higher ANGPT2 concentrations were observed in the plasma, lungs, kidneys, and eyes (P=0.0212, P<0.001, P=0.0815, and P=0.0215, respectively). Additionally, the Schlemm canal was larger in MT mice compared with WT mice (P=0.0430). The rs76020419 minor allele (T) is associated with increased levels of ANGPT2 and a larger Schlemm canal in mice. These findings suggest a potential protective mechanism in glaucoma.

A Novel TEK::GAB2 Gene Fusion in Pediatric Angiosarcoma of Pelvic soft Tissue: A Case Report and Literature Review.

Pediatric angiosarcoma of soft tissue, an extremely rare entity, remains poorly understood from a genetic standpoint. Herein, we present the case of a previously healthy 17-year-old girl with acute left hip pain. Subsequent magnetic resonance imaging revealed a 21.8 cm left pelvic sidewall mass with heterogeneous enhancement and multiple lung nodules. Biopsy of the tumor showed an infiltrative, hemorrhagic neoplasm composed primarily of atypical spindle to epithelioid cells. Focal vasoformative architecture was appreciated. Immunohistochemically, the tumor cells were strongly positive for CD31, ERG, and FLI-1, supporting the diagnosis of angiosarcoma. Genetic analysis identified a novel TEK::GAB2 gene fusion. TEK belongs to the angiopoietin receptor family, and its fusion with GAB2 is predicted to mediate tumorigenesis. This report expands the current knowledge on the spectrum of gene rearrangements of angiosarcoma.

Fluid bolus increases plasma hyaluronan concentration compared to follow-up strategy without a bolus in oliguric intensive care unit patients

Fluid therapy is a fundamental part of supportive therapy in critical care. However, it is also a suspected risk for endothelial glycocalyx degradation which is associated with poor clinical outcomes. This secondary analysis of RESPONSE randomized trial compares the effect of follow-up strategy (FU) on endothelial biomarkers to that of 500 ml crystalloid fluid bolus (FB) in oliguric, hemodynamically optimized intensive care unit (ICU) patients. 130 adult subjects were enrolled in two Finnish ICUs from January 2017 to November 2020. Blood and urine samples of 63 patients in FU group and 67 patients in FB group were collected before and after the intervention and analyzed using enzyme-linked immunosorbent assays. Single fluid bolus, given after median of 3887 ml (interquartile range 2842; 5359 ml) resuscitation fluids in the preceding 24 h, increased plasma hyaluronan concentration compared to the follow-up strategy (difference in medians 29.2 ng/ml with 95% CI [14.5ng/ml; 55.5ng/ml], P < 0.001). No treatment effect was detected in the plasma levels of syndecan-1, , angiopoietin-2, angiopoietin receptors Tie2 and Tie1, or in soluble thrombomodulin in the adjusted median regression analysis. The increase in hyaluronan was independent of its simultaneous renal clearance but correlated moderately with the increase in endothelium-specific Tie1. The follow-up strategy did not show consistent endothelium-sparing effect but protected against hyaluronan increase. The mechanisms and consequences of hyaluronan fluctuations need further clarification. Trial registration: clinicaltrials.gov, NCT02860572. Registered 1 August 2016, https://www.clinicaltrials.gov/study/NCT02860572?term=NCT02860572&rank=1

Unveiling novel serum biomarkers in intrahepatic cholangiocarcinoma: a pilot proteomic exploration.

Recent advancements in proteomics have shown promise in identifying biomarkers for various cancers. Our study is the first to compare the serum proteomes of intrahepatic cholangiocarcinoma (iCCA) with cirrhosis (CIR), primary sclerosing cholangitis (PSC), and hepatocellular carcinoma (HCC), aiming to identify a proteomic signature that can effectively distinguish among these conditions. Utilizing high-throughput mass spectrometry on serum samples, we identified 845 proteins, of which 646 were suitable for further analysis. Unique clustering patterns were observed among the five groups, with significant proteomic differences. Our key findings include: S100 calcium-binding protein A9 (S100A9) and haptoglobin (HP) were more abundant in iCCA, while intercellular adhesion molecule 2 (ICAM2) was higher in HCC. Serum amyloid A1 (SAA1) and A4 (SAA4) emerged as potential biomarkers, with SAA1 significantly different in the iCCA vs healthy controls (HC) comparison, and SAA4 in the HCC vs HC comparison. Elevated levels of vascular cell adhesion molecule 1 (VCAM-1) in HCC suggested its potential as a differentiation and diagnostic marker. Angiopoietin-1 receptor (TEK) also showed discriminatory and diagnostic potential in HCC. ELISA validation corroborated mass spectrometry findings. Our study underscores the potential of proteomic profiling in distinguishing iCCA from other liver conditions and highlights the need for further validation to establish robust diagnostic biomarkers.

Targeting the Tie-2 Receptor With Faricimab in Central Serous Chorioretinopathy: A Case Series Motivated by a Genetic Finding

PurposeTo investigate the effects of faricimab, a bispecific antibody targeting VEGF and Ang-2 (thus increasing Tie-2 activity), in patients with CSC based on a recent genetic study that implicated Tie-2 signaling in CSC pathophysiology. DesignA retrospective interventional multicenter case series. MethodsWe included patients with chronic CSC (persistent or recurrent SRF for ≥6 months) who received at least one faricimab 6mg injection between January 1 2022 and April 1 2024. Study sites included Massachusetts Eye and Ear and University of California San Francisco. Patients with evidence of a choroidal neovascular membrane on color photos, optical coherence tomography (OCT) and/or fluorescein angiography were excluded. 16 eyes (15 patients) met the inclusion criteria. The median central macular thickness at each visit from 52 weeks before to 52 weeks after the first faricimab injection was calculated using automated Heidelberg Spectralis ETDRS subfield measurements. ResultsPrior to treatment with faricimab, CSC had been diagnosed a median of 4.1 years (range 0.9–8) earlier and SRF (and intraretinal fluid [IRF] in a subset) had been continuously present for a median of 30 weeks (range 9–257). Decreases in macular thickness were observed in 14/16 eyes after the first faricimab injection and in 14/16 eyes in the full follow-up period compared with prior, 10 of which experienced complete resolution of SRF following the start of the first series of injections at a median of 4 weeks (range 2—25). One eye worsened after the second injection. The median improvement in macular thickness was 40μm [range -3 to 89.5] (P=0.0007). Upon review of OCT images, reductions in macular thickness were consistent with reductions in SRF and/or IRF. Visual acuity improved by 2 lines or more in 6/16 eyes. ConclusionsIn a retrospective case series of patients with chronic CSC and longstanding SRF, we observed improvement in macular thickness after intravitreal faricimab. While the small number of patients and variable natural history of CSC preclude definitive conclusions, a randomized controlled trial seems warranted.

VEPTP inhibition with an extracellular domain targeting antibody did not restore albuminuria in a mouse model of diabetic kidney disease.

Diabetic kidney disease (DKD) is the leading cause of end-stage kidney disease. DKD is a heterogeneous disease with complex pathophysiology where early endothelial dysfunction is associated with disease progression. The Tie2 receptor and Angiopoietin 1 and 2 ligands are critical for maintaining endothelial cell permeability and integrity. Tie2 signaling is negatively regulated by the endothelial specific transmembrane receptor Vascular Endothelial Protein Tyrosine Phosphatase (VEPTP). Genetic deletion of VEPTP protects from hypertension and diabetes induced renal injury in a mouse model of DKD. Here, we show that VEPTP inhibition with an extracellular domain targeting VEPTP antibody induced Tie2 phosphorylation and improved VEGF-A induced vascular permeability both invitro and invivo. Treatment with the VEPTP blocking antibody decreased the renal expression of endothelial activation markers (Angpt2, Edn1, and Icam1) but failed to improve kidney function in db/db uninephrectomized ReninAAV DKD mice.

Abstract P184: Increased Granulopoiesis, Kidney Neutrophil Infiltration, and Renal Damage in a Mouse Model of Psoriasis

Psoriasis is an autoimmune skin disease marked by T cell-mediated hyperproliferation of epidermal keratinocytes. Most psoriasis-related morbidity and mortality stems from an elevated risk of renal and cardiovascular disease, but mechanisms remain unclear. Although neutrophils are not directly targeted with current psoriasis therapies, neutrophils are increased in the skin and circulation of these patients. We hypothesized that excess skin inflammation in psoriasis drives increased G-CSF-dependent granulopoiesis leading to renal neutrophil infiltration and dysfunction. Employing a KC-Tie2 mouse model of psoriasis with keratinocyte-specific overexpression of the angiopoietin receptor Tie2 (KC-Tie2), we observed 2-3-fold elevations in multiple indices of renal damage including urine albumin, urine NGAL, and glomerulosclerosis in KC-Tie2 mice compared to littermate controls (all P&lt;0.05). KC-Tie2 mice also demonstrated 13 mm Hg higher daytime systolic blood pressure compared to littermate controls (P&lt;0.05). Using flow cytometry, we found that KC-Tie2 mice had an increase not only in cutaneous neutrophils but also an approximate 10-fold increase in renal neutrophils (P&lt;0.001). KC-Tie2 kidneys also exhibited increased vital and suicidal neutrophil extracellular trap (NET) formation, representing a potential mechanism for the observed renal damage. Interestingly, the increase in renal neutrophils was selective relative to other major immune cell populations such as dendritic cells, lymphocytes, monocytes, and macrophages. Similar selective increases in neutrophils were noted in the circulation, aorta, and spleen. We thus examined neutrophil production in the bone marrow, and observed significant increases in immature Ly6g low and Ly6g intermediate neutrophils (P&lt;0.0001) as well as total Ly6g + neutrophils (P&lt;0.05), with no change in mature Ly6g high neutrophils in the bone marrow of KC-Tie2 mice. These findings are consistent with increased granulopoiesis, prompting an examination of the key mediator of granulopoiesis, granulocyte colony-stimulating factor (G-CSF). KC-Tie2 mice exhibited marked increases in G-CSF in both the skin and plasma (P&lt;.001 for both). In conclusion, our findings demonstrate increased renal damage in a mouse model of psoriasis, concomitant with increases in granulopoiesis and total and NETotic renal neutrophils, potentially unveiling a novel link between skin inflammation and renal damage via enhanced G-CSF-mediated granulopoiesis.

Novel bibenzyl compound Ae exhibits anti-agiogenic activity in HUVECs in vitro and zebrafish in vivo

The inhibition of angiogenesis has been considered as an attractive method for the discovery of potential anti-cancer drugs. Herein, we report our new synthesized bibenzyl compound Ae had potent anti-angiogenic activity(the lowest effective concentration is to 0.62–1.25 μM) in zebrafish in vivo and showed a concentration-dependent inhibition of inter-segmental blood vessels (ISVs) compared to control. Further, Ae exhibited the obvious inhibitory activity of proliferation, migration, invasion and tube formation in HUVEC cells in vitro. Moreover, qRT-PCR analysis revealed that the anti-angiogenic activity of compound Ae is connected with the ang-2, tek in ANGPT-TEK pathway and the kdr, kdrl signaling axle in VEGF-VEGFR pathway. Molecular docking studies revealed that compound Ae had an interaction with the angiopoietin-2 receptor(TEK) and VEGFR2. Additionally, analysis of the ADMET prediction data indicated that compound Ae possessed favorable physicochemical properties, drug-likeness, and synthetic accessibility. In conclusion, compound Ae had remarkable anti-angiogenic activity and could be served as an candidate for cancer therapy.

Treatment of vascular dementia in female rats with AV-001, an Angiopoietin-1 mimetic peptide, improves cognitive function.

Vascular dementia (VaD) is a complex neurodegenerative disorder. We previously found that treatment of VaD in middle-aged male rats subjected to multiple microinfarction (MMI) with AV-001, a Tie2 receptor agonist, significantly improves cognitive function. Age and sex affect the development and response of VaD to therapeutic intervention. Thus, the present study investigated the therapeutic effect of AV-001 on VaD in aged female rats subjected to MMI. Female 18-month-old Wistar rats were subjected to MMI by injecting either 1,000 (low dose, LD-MMI) or 6,000 (high dose, HD-MMI) cholesterol crystals of size 70-100 μm into the right internal carotid artery. AV-001 (1 μg/Kg, i.p.) was administered once daily after MMI for 1 month, with treatment initiated 1 day after MMI. A battery of behavioral tests to examine sensorimotor and cognitive functions was performed at 21-28 days after MMI. All rats were sacrificed at 1 month after MMI. Aged female rats subjected to LD-MMI exhibit severe neurological deficits, memory impairment, and significant white matter (WM) and oligodendrogenesis injury in the corpus callosum compared with control rats. HD-MMI in aged female rats induces significant anxiety- and depression-like behaviors, which were not detected in LD-MMI aged female rats. Also, HD-MMI induces significantly increased WM injury compared to LD-MMI. AV-001 treatment of LD-MMI and HD-MMI increases oligodendrogenesis, myelin and axon density in the corpus callosum and striatal WM bundles, promotes WM integrity and attenuates neurological and cognitive deficits. Additionally, both LD-MMI and HD-MMI rats exhibit a significant increase, while AV-001 significantly decreases the levels of inflammatory factors in the cerebrospinal fluid (CSF). MMI reduces oligodendrogenesis, and induces demyelination, axonal injury and WM injury, and causes memory impairment, while HD-MMI induces increased WM injury and further depression-like behaviors compared to LD-MMI rats. AV-001 has a therapeutic effect on aged female rats with MMI by reducing WM damage and improving neuro-cognitive outcomes.