HIF-1α-dependent regulation of angiogenic factor expression in Müller cells by mechanical stimulation.

Mechanical stress regulates various biological processes in cells, tissues, and organs as well as contributes to the pathogenesis of various diseases. The retina is subjected to mechanical stress imposed by intraocular pressure as well as by retinal hemorrhage and edema. Responses to mechanical stress have been studied in retinal pigment epithelial cells and Müller cells of the retina, with the former cells having been found to undergo a stress-induced increase in the expression of vascular endothelial growth factor (VEGF), which plays a key role in physiological and pathological angiogenesis in the retina. We here examined the effects of stretch stimulation on the expression of angiogenic factors in cultured human Müller cells. Reverse transcription and quantitative PCR analysis revealed that expression of the VEGF-A gene was increased by such stimulation in Müller cells, whereas that of the angiopoietin 1 gene was decreased. An enzyme-linked immunosorbent assay showed that stretch stimulation also increased VEGF secretion from these cells. Expression of the transcription factor HIF-1α (hypoxia-inducible factor-1α) was increased at both mRNA and protein levels by stretch stimulation, and the HIF-1α inhibitor CAY10585 prevented the effects of mechanical stress on VEGF-A gene expression and VEGF secretion. Furthermore, RNA-sequencing analysis showed that the expression of angiogenesis-related pathway genes was upregulated by stretch stimulation. Our results thus suggest that mechanical stress induces VEGF production in Müller cells in a manner dependent on HIF-1α, and that HIF-1α is therefore a potential therapeutic target for conditions such as diabetic retinopathy, age-related macular degeneration, and retinal vein occlusion.

Platelet Angiopoietin-1 Protects Against Murine Models of Tumor Metastasis.

BACKGROUND: In addition to their fundamental roles in preserving vascular integrity, platelets also contribute to tumor angiogenesis and metastasis. However, despite being a reservoir for angiogenic and metastatic cytokines, platelets also harbor negative regulators of tumor progression. Angpt1 (angiopoietin-1) is a cytokine essential for developmental angiogenesis that also protects against tumor cell metastasis through an undefined mechanism. Although activated platelets release Angpt1 from α-granules into circulation, the contributions of platelet Angpt1 to tumor growth, angiogenesis, and metastasis have not been investigated. METHODS: Using cytokine arrays and ELISAs, we first compared platelet Angpt1 levels in breast and melanoma mouse tumor models to tumor-free controls. We then assessed tumor growth and metastasis in mice lacking megakaryocyte and platelet Angpt1 (Angpt1Plt KO). The spontaneous metastasis of mammary-injected tumor cells to the lungs was quantified using RT-PCR (reverse transcription-polymerase chain reaction). The lung colonization of intravenously injected tumor cells and tumor cell extravasation were determined using fluorescent microscopy and flow cytometry. RESULTS: Platelet Angpt1 is selectively upregulated in the PyMT (polyoma middle tumor antigen) breast cancer mouse model, and platelets are the principal source of Angpt1 in blood circulation. While primary tumor growth and angiogenesis were unaffected, Angpt1Plt KO mice had both increased spontaneous lung metastasis and tumor cell lung colonization following mammary or intravenous injection, respectively. Although platelet Angpt1 did not affect initial tumor cell entrapment in the lungs, Angpt1Plt KO mice had increased tumor cell retention and extravasation. Serum from Angpt1Plt KO mice increased endothelial permeability and reduced VE (vascular endothelial)-cadherin expression at endothelial junctions compared with serum from control mice (Angpt1WT). CONCLUSIONS: Platelets provide an intravascular source of Angpt1 that restrains tumor metastasis by preserving the lung microvasculature to limit tumor cell extravasation.

Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma.

BACKGROUND: It has been proposed that anti-angiogenesis therapy could induce tumor "vascular normalization" and further enhance the efficacy of chemotherapy, radiotherapy, target therapy, and immunotherapy for nearly twenty years. However, the detailed molecular mechanism of this phenomenon is still obscure. METHOD: Overexpression and knockout of CCL28 in human lung adenocarcinoma cell line A549 and murine lung adenocarcinoma cell line LLC, respectively, were utilized to establish mouse models. Single-cell sequencing was performed to analyze the proportion of different cell clusters and metabolic changes in the tumor microenvironment (TME). Immunofluorescence and multiplex immunohistochemistry were conducted in murine tumor tissues and clinical biopsy samples to assess the percentage of pericytes coverage. Primary pericytes were isolated from lung adenocarcinoma tumor tissues using magnetic-activated cell sorting (MACS). These pericytes were then treated with recombinant human CCL28 protein, followed by transwell migration assays and RNA sequencing analysis. Changes in the secretome and metabolome were examined, and verification of retinoic acid metabolism alterations in pericytes was conducted using quantitative real-time PCR, western blotting, and LC-MS technology. Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) was employed to validate the transcriptional regulatory ability and affinity of RXRα to specific sites at the ANGPT1 promoter. RESULTS: Our study showed that after undergoing anti-angiogenesis treatment, the tumor exhibited a state of ischemia and hypoxia, leading to an upregulation in the expression of CCL28 in hypoxic lung adenocarcinoma cells by the hypoxia-sensitive transcription factor CEBPB. Increased CCL28 could promote tumor vascular normalization through recruiting and metabolic reprogramming pericytes in the tumor microenvironment. Mechanistically, CCL28 modified the retinoic acid (RA) metabolism and increased ANGPT1 expression via RXRα in pericytes, thereby enhancing the stability of endothelial cells. CONCLUSION: We reported the details of the molecular mechanisms of "vascular normalization" after anti-angiogenesis therapy for the first time. Our work might provide a prospective molecular marker for guiding the clinical arrangement of combination therapy between anti-angiogenesis treatment and other therapies.

Pseudomonas aeruginosa N-3-Oxododecanoyl Homoserine Lactone Disrupts Endothelial Integrity by Activating the Angiopoietin-Tie System.

The activation of the angiopoietin (Angpt)-Tie system is linked to endothelial dysfunction during sepsis. Bacterial quorum-sensing molecules function as pathogen-associated molecular patterns. However, their impact on the endothelium and the Angpt-Tie system remains unclear. Therefore, this study investigated whether treatment with N-3-oxododecanoyl homoserine lactone (3OC12-HSL), a quorum-sensing molecule derived from Pseudomonas aeruginosa, impaired endothelial function in human umbilical vein endothelial cells. 3OC12-HSL treatment impaired tube formation even at sublethal concentrations, and immunocytochemistry analysis revealed that it seemed to reduce vascular endothelial-cadherin expression at the cell-cell interface. Upon assessing the mRNA expression patterns of genes associated with the Angpt-Tie axis, the expressions of Angpt2, Forkhead box protein O1, Tie1, and vascular endothelial growth factor 2 were found to be upregulated in the 3OC12-HSL-treated cells. Moreover, western blot analysis revealed that 3OC12-HSL treatment increased Angpt2 expression. A co-immunoprecipitation assay was conducted to assess the effect of 3OC12-HSL on the IQ motif containing GTPase activating protein 1 (IQGAP1) and Rac1 complex and the interaction between these proteins was consistently maintained regardless of 3OC12-HSL treatment. Next, recombinant human (rh)-Angpt1 was added to assess whether it modulated the effects of 3OC12-HSL treatment. rh-Angpt1 addition increased cellular viability, improved endothelial function, and reversed the overall patterns of mRNA and protein expression in endothelial cells treated with 3OC12-HSL. Additionally, it was related to the increased expression of phospho-Akt and the IQGAP1 and Rac1 complex. Collectively, our findings indicated that 3OC12-HSL from Pseudomonas aeruginosa can impair endothelial integrity via the activation of the Angpt-Tie axis, which appeared to be reversed by rh-Angpt1 treatment.

Angpt1 binding to Tie1 regulates the signaling required for lymphatic vessel development in zebrafish.

Development of the vascular system is regulated by multiple signaling pathways mediated by receptor tyrosine kinases. Among them, angiopoietin (Ang)/Tie signaling regulates lymphatic and blood vessel development in mammals. Of the two Tie receptors, Tie2 is well known as a key mediator of Ang/Tie signaling, but, unexpectedly, recent studies have revealed that the Tie2 locus has been lost in many vertebrate species, whereas the Tie1 gene is more commonly present. However, Tie1-driven signaling pathways, including ligands and cellular functions, are not well understood. Here, we performed comprehensive mutant analyses of angiopoietins and Tie receptors in zebrafish and found that only angpt1 and tie1 mutants show defects in trunk lymphatic vessel development. Among zebrafish angiopoietins, only Angpt1 binds to Tie1 as a ligand. We indirectly monitored Ang1/Tie1 signaling and detected Tie1 activation in sprouting endothelial cells, where Tie1 inhibits nuclear import of EGFP-Foxo1a. Angpt1/Tie1 signaling functions in endothelial cell migration and proliferation, and in lymphatic specification during early lymphangiogenesis, at least in part by modulating Vegfc/Vegfr3 signaling. Thus, we show that Angpt1/Tie1 signaling constitutes an essential signaling pathway for lymphatic development in zebrafish.

Differential expression of angiogenesis-related genes 'VEGF' and 'angiopoietin-1' in metastatic and EMAST-positive colorectal cancer patients.

Abnormal angiogenesis leads to tumor progression and metastasis in colorectal cancer (CRC). This study aimed to elucidate the association between angiogenesis-related genes, including VEGF-A, ANGPT-1, and ANGPT-2 with both metastatic and microsatellite alterations at selected tetranucleotide repeats (EMAST) subtypes of CRC. We conducted a thorough assessment of the ANGPT-1, ANGPT-2, and VEGF-A gene expression utilizing publicly available RNA sequencing and microarray datasets. Then, the experimental validation was performed in 122 CRC patients, considering their disease metastasis and EMAST+/- profile by using reverse transcription polymerase chain reaction (RT-PCR). Subsequently, a competing endogenous RNA (ceRNA) network associated with these angiogenesis-related genes was constructed and analyzed. The expression level of VEGF-A and ANGPT-2 genes were significantly higher in tumor tissues as compared with normal adjacent tissues (P-value < 0.001). Nevertheless, ANGPT-1 had a significantly lower expression in tumor samples than in normal colon tissue (P-value < 0.01). We identified a significantly increased VEGF-A (P-value = 0.002) and decreased ANGPT-1 (P-value = 0.04) expression in EMAST+ colorectal tumors. Regarding metastasis, a significantly increased VEGF-A and ANGPT-2 expression (P-value = 0.001) and decreased ANGPT-1 expression (P-value < 0.05) were established in metastatic CRC patients. Remarkably, co-expression analysis also showed a strong correlation between ANGPT-2 and VEGF-A gene expressions. The ceRNA network was constructed by ANGPT-1, ANGPT-2, VEGF-A, and experimentally validated miRNAs (hsa-miR-190a-3p, hsa-miR-374c-5p, hsa-miR-452-5p, and hsa-miR-889-3p), lncRNAs (AFAP1-AS1, KCNQ1OT1 and MALAT1), and TFs (Sp1, E2F1, and STAT3). Network analysis revealed that colorectal cancer is amongst the 82 significant pathways. We demonstrated a significant differential expression of VEGF-A and ANGPT-1 in colorectal cancer patients exhibiting the EMAST+ phenotype. This finding provides novel insights into the molecular pathogenesis of colorectal cancer, specifically in EMAST subtypes. Yet, the generalization of in silico findings to EMAST+ colorectal cancer warrants future experimental investigations. In the end, this study proposes that the EMAST biomarker could serve as an additional perspective on CMS4 biology which is well-defined by activated angiogenesis and worse overall survival.

MiR-221-5p regulates blood-brain barrier dysfunction through the angiopoietin-1/-2/Tie-2 signaling axis after subarachnoid hemorrhage.

AIM: To explore the potential role of microRNA miR-221-5p on the angiopoietin-1 (Ang-1)/Ang-2/Tie-2 signaling axis after subarachnoid hemorrhage (SAH) in a rat model. METHODS: Aspects of the rat's behavior were measured using the Kaoutzanis scoring system to test neurological responses. This included feeding behavior, body contraction, motor, and eye-opening responses. Brain sections were studied using transmission electron microscopy and Evans blue extravasation. Levels of Ang-1, Ang-2, and Tie-2 were determined by Western blot, while miR-221-5p was quantified using stem-loop real-time quantitative PCR (RT-qPCR). RESULTS: The SAH group responded worse to the neurological response test than the sham-operated group. The intercellular space was widened in the SAH group, but not in the sham-operated group. Evans blue dye leaked significantly more into brain tissue cells of the SAH group. Stem-loop qRT-PCR showed elevated miR-221-5p levels. Additionally, Ang-1 and Tie-2 were reduced but Ang-2 expression was increased after SAH. This led to a significant reduction of the Ang-1/Ang-2 ratio in the brain tissue, which was associated with the destruction of the blood-brain barrier. CONCLUSION: The data indicate that miR-221-5p might regulate blood-brain barrier dysfunction through the Ang-1/Ang-2/Tie-2 signaling axis, suggesting that it should be further investigated as a potential novel biomarker.

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.

Detrimental effects of PCSK9 loss-of-function in the pediatric host response to sepsis are mediated through independent influence on Angiopoietin-1.

BACKGROUND: Sepsis is associated with significant mortality. Yet, there are no efficacious therapies beyond antibiotics. PCSK9 loss-of-function (LOF) and inhibition, through enhanced low-density lipoprotein receptor (LDLR) mediated endotoxin clearance, holds promise as a potential therapeutic approach among adults. In contrast, we have previously demonstrated higher mortality in the juvenile host. Given the potential pleiotropic effects of PCSK9 on the endothelium, beyond canonical effects on serum lipoproteins, both of which may influence sepsis outcomes, we sought to test the influence of PCSK9 LOF genotype on endothelial dysfunction. METHODS: Secondary analyses of a prospective observational cohort of pediatric septic shock. Genetic variants of PCSK9 and LDLR genes, serum PCSK9, and lipoprotein concentrations were determined previously. Endothelial dysfunction markers were measured in day 1 serum. We conducted multivariable linear regression to test the influence of PCSK9 LOF genotype on endothelial markers, adjusted for age, complicated course, and low- and high-density lipoproteins (LDL and HDL). Causal mediation analyses to test impact of select endothelial markers on the association between PCSK9 LOF genotype and mortality. Juvenile Pcsk9 null and wildtype mice were subject to cecal slurry sepsis and endothelial markers were quantified. RESULTS: A total of 474 patients were included. PCSK9 LOF was associated with several markers of endothelial dysfunction, with strengthening of associations after exclusion of those homozygous for the rs688 LDLR variant that renders it insensitive to PCSK9. Serum PCSK9 was not correlated with endothelial dysfunction. PCSK9 LOF influenced concentrations of Angiopoietin-1 (Angpt-1) upon adjusting for potential confounders including lipoprotein concentrations, with false discovery adjusted p value of 0.042 and 0.013 for models that included LDL and HDL, respectively. Causal mediation analysis demonstrated that the effect of PCSK9 LOF on mortality was mediated by Angpt-1 (p = 0.0008). Murine data corroborated these results with lower Angpt-1 and higher soluble thrombomodulin among knockout mice with sepsis relative to the wildtype. CONCLUSIONS: We present genetic and biomarker association data that suggest a potential direct role of the PCSK9-LDLR pathway on Angpt-1 in the developing host with septic shock and warrant external validation. Further, mechanistic studies on the role of PCSK9-LDLR pathway on vascular homeostasis may lead to the development of pediatric-specific sepsis therapies.

The expression analyses of GSK3B, VEGF, ANG1, and ANG2 in human brain microvascular endothelial cells treated with the synthetic cannabinoid XLR-11.

The endocannabinoid system receptors, cannabinoid receptors type-1 (CBR-1) and -2 (CBR-2), are implicated in several behavioral and cognitive processes. Many studies have indicated a correlation between cannabinoid receptors and angiogenesis. The current study aims to reveal the possible molecular signaling involved in brain angiogenesis induced by the activation of CBR-1 and CBR-2. We investigated whether the synthetic cannabinoid XLR-11, an agonist of CBR-1 and CBR-2, influences the mRNA and protein expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (ANG1) and -2 (ANG2) in human brain microvascular endothelial cells (hBMVEs). Furthermore, we determined the phosphorylation of glycogen synthase kinase 3 beta (GSK3B) expression. Treatment of hBMVEs cells with XLR-11 elevated the mRNA levels of VEGF, ANG1, and ANG2. The secretion of these proangiogenic factors was increased in the media. Furthermore, the intracellular expression of VEGF, ANG1, ANG2, and GSK3B was significantly increased. This current research provides a new possible approach by targeting the cannabinoid receptors to control and regulate brain angiogenesis for treating a variety of angiogenesis-related diseases. This could be achived by using different agonists or antagonists of the cannabinoid receptors based on the nature of the diseases.

Angiopoietin-1 promotes triple-negative breast cancer cell proliferation by upregulating carboxypeptidase A4.

Angiopoietin-1 (ANG1) is a pro-angiogenic regulator that contributes to the progression of solid tumors by stimulating the proliferation, migration and tube formation of vascular endothelial cells, as well as the renewal and stability of blood vessels. However, the functions and mechanisms of ANG1 in triple-negative breast cancer (TNBC) are unclear. The clinical sample database shows that a higher level of ANG1 in TNBC is associated with poor prognosis compared to non-TNBC. In addition, knockdown of ANG1 inhibits TNBC cell proliferation and induces cell cycle G1 phase arrest and apoptosis. Overexpression of ANG1 promotes tumor growth in nude mice. Mechanistically, ANG1 promotes TNBC by upregulating carboxypeptidase A4 (CPA4) expression. Overall, the ANG1-CPA4 axis can be a therapeutic target for TNBC.

Plasmid containing VEGF-165 and ANG-1 dual genes packaged with fibroin-modified PEI to promote the regeneration of vascular network and dermal tissue.

Reducing the cytotoxicity of cationic polymers is the major issue to their use as a gene delivery carrier. In this study, plasmids containing encoding vascular endothelial cell growth factor 165 and angiopoietin-1 were packaged with the conjugates of cationic fibroin (CSF) and polyethylenimine (PEI), instead of packaging pDNA with PEI alone, to prepare nanocomplexes (CSF+PEI)/pDNA. The complexes were loaded into a silk fibroin scaffold to enhance its function to induce microvascular network generation and dermal tissue regeneration. The results of transfecting EA.hy926 cells with the complexes in vitro showed that (CSF+PEI)/pDNA had a stronger transfection ability than PEI/pDNA. Importantly, compared with PEI as the gene carrier alone, the cell viability was significantly increased and the cytotoxicity was effectively reduced after the conjugate of CSF and PEI was used as the gene carrier. The results of angiogenesis in chick embryo chorioallantoic membranes showed that compared with scaffolds loaded with PEI/pDNA, the neovascularization ratio in scaffolds loaded with (CSF+PEI)/pDNA was significantly increased. In vivo experimental results of scaffolds implantation for full-thickness skin defects in SD rats showed that, compared with loading PEI/pDNA complex, loading (CSF+PEI)/pDNA complex in the scaffold more effectively promoted the formation of vascular network in the scaffold and accelerated the regeneration of dermal tissue. The gene delivery system established in this study has application potential not only in the regeneration of vascular-containing tissues, but also in tumor gene therapy.

Intracellular angiopoietin-1 promotes TKI-resistance via activation of JAK/STAT5 pathway in chronic myeloid leukemia.

Drug resistance from BCR-ABL tyrosine kinase inhibitors (TKIs) and other chemotherapeutics results in treatment failure and disease progression in chronic myeloid leukemia (CML). However, the mechanism is still uncertain. In this study, we investigated the role of angiopoietin-1 (ANG-1) as a potential prognostic factor for drug resistance in CML. Both intracellular and secretory ANG-1 (iANG-1 and sANG-1) were overexpressed in multidrug-resistant CML samples. The IC50 value was higher in primary CD34+ CD38- cells with more ANG-1. Silencing ANG-1significantly sensitized three TKI-resistant CML cell lines to imatinib (IM) while recombinant human ANG-1 failed to retain cell survival in vitro. This indicated the important role of iANG-1 as opposed to sANG-1 in CML drug resistance. Moreover, a similar effect was observed in xenograft mice models bearing ANG-1-silenced CML cells. Subsequently, pathway analysis and protein validation experiments showed activation of the JAK/STAT pathway and augmentation of STAT5a phosphorylation in ANG-1 restored CML cells. Upstream Src phosphorylation, which plays a crucial role in CML drug resistance, was also upregulated as a key event in iANG-1-related JAK/STAT pathway activation. In conclusion, our study elucidated a new BCR-ABL independent molecular mechanism induced by intracytoplasmic ANG-1 overexpression as a potential strategy for overcoming CML resistance.

Local and Systemic Overexpression of COMP-Ang1 Induces Ang1/Tie2-Related Thrombocytopenia and SDF-1/CXCR4-Dependent Anemia.

While supplemental angiopoietin-1 (Ang1) improves hematopoiesis, excessive Ang1 induces bone marrow (BM) impairment, hematopoietic stem cell (HSC) senescence, and erythropoietic defect. Here, we examined how excessive Ang1 disturbs hematopoiesis and explored whether hematopoietic defects were related to its level using K14-Cre;c-Ang1 and Col2.3-Cre;c-Ang1 transgenic mice that systemically and locally overexpress cartilage oligomeric matrix protein-Ang1, respectively. We also investigated the impacts of Tie2 inhibitor and AMD3100 on hematopoietic development. Transgenic mice exhibited excessive angiogenic phenotypes, but K14-Cre;c-Ang1 mice showed more severe defects in growth and life span with higher presence of Ang1 compared with Col2.3-Cre;c-Ang1 mice. Dissimilar to K14-Cre;c-Ang1 mice, Col2.3-Cre;c-Ang1 mice did not show impaired BM retention or senescence of HSCs, erythropoietic defect, or disruption of the stromal cell-derived factor 1 (SDF-1)/CXCR4 axis. However, these mice exhibited a defect in platelet production depending on the expression of Tie2 and globin transcription factor 1 (GATA-1), but not GATA-2, in megakaryocyte progenitor (MP) cells. Treatment with Tie2 inhibitor recovered GATA-1 expression in MP cells and platelet production without changes in circulating RBC in transgenic mice. Consecutive AMD3100 administration not only induced irrecoverable senescence of HSCs but also suppressed formation of RBC, but not platelets, via correlated decreases in number of erythroblasts and their GATA-1 expression in B6 mice. Our results indicate that genetic overexpression of Ang1 impairs hematopoietic development depending on its level, in which megakaryopoiesis is preferentially impaired via activation of Ang1/Tie2 signaling, whereas erythropoietic defect is orchestrated by HSC senescence, inflammation, and disruption of the SDF-1/CXCR4 axis.

Multi-Scalar Data Integration Links Glomerular Angiopoietin-Tie Signaling Pathway Activation With Progression of Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is the leading cause of end-stage kidney disease (ESKD). Prognostic biomarkers reflective of underlying molecular mechanisms are critically needed for effective management of DKD. A three-marker panel was derived from a proteomics analysis of plasma samples by an unbiased machine learning approach from participants (N = 58) in the Clinical Phenotyping and Resource Biobank study. In combination with standard clinical parameters, this panel improved prediction of the composite outcome of ESKD or a 40% decline in glomerular filtration rate. The panel was validated in an independent group (N = 68), who also had kidney transcriptomic profiles. One marker, plasma angiopoietin 2 (ANGPT2), was significantly associated with outcomes in cohorts from the Cardiovascular Health Study (N = 3,183) and the Chinese Cohort Study of Chronic Kidney Disease (N = 210). Glomerular transcriptional angiopoietin/Tie (ANG-TIE) pathway scores, derived from the expression of 154 ANG-TIE signaling mediators, correlated positively with plasma ANGPT2 levels and kidney outcomes. Higher receptor expression in glomeruli and higher ANG-TIE pathway scores in endothelial cells corroborated potential functional effects in the kidney from elevated plasma ANGPT2 levels. Our work suggests that ANGPT2 is a promising prognostic endothelial biomarker with likely functional impact on glomerular pathogenesis in DKD.

Cooperation of Angiopoietin-2 and Angiopoietin-4 in Schlemm's Canal Maintenance.

Purpose: Defects in the iridocorneal angle tissues, including the trabecular meshwork (TM) and Schlemm's canal (SC), impair aqueous humor flow and increase the intraocular pressure (IOP), eventually resulting in glaucoma. Activation of endothelial tyrosine kinase receptor Tie2 by angiopoietin-1 (Angpt1) has been demonstrated to be essential for SC formation, but roles of the other two Tie2 ligands, Angpt2 and Angpt4, have been controversial or not yet characterized, respectively. Methods: Angpt4 expression was investigated using genetic cell fate mapping and reporter mice. Congenital deletion of Angpt2 and Angpt4 and tamoxifen-inducible deletion of Angpt1 in mice were used to study the effects of Angpt4 deletion alone and in combination with the other angiopoietins. SC morphology was examined with immunofluorescent staining. IOP measurements, electron microscopy, and histologic evaluation were used to study glaucomatous changes. Results: Angpt4 was postnatally expressed in the TM. While Angpt4 deletion alone did not affect SC and Angpt4 deletion did not aggravate Angpt1 deletion phenotype, absence of Angpt4 combined with Angpt2 deletion had detrimental effects on SC morphology in adult mice. Consequently, Angpt2-/-;Angpt4-/- mice displayed glaucomatous changes in the eye. Mice with Angpt2 deletion alone showed only moderate SC defects, but Angpt2 was necessary for proper limbal vasculature development. Mechanistically, analysis of Tie2 phosphorylation suggested that Angpt2 and Angpt4 cooperate as agonistic Tie2 ligands in maintaining SC integrity. Conclusions: Our results indicated an additive effect of Angpt4 in SC maintenance and Tie2 activation and a spatiotemporally regulated interplay between the angiopoietins in the mouse iridocorneal angle.

Pro-angiogenic activity of isofuran.

Isofurans (IsoFs) are a series of novel discovered lipid peroxidation products. This study focused on the investigation of the angiogenic property of IsoF. MTT stain assay indicated that 1 µm IsoF had the most bioactivity in rat brain endothelial cells (RBECs). IsoF significantly promoted cellular proliferation and migration and remarkably decreased staurosporine-induced apoptosis by TUNEL assay in the RBECs. It successfully up-regulated rat aortic vascularization and choroid explant sprouting, extracellular regulated protein kinases (ERK)1/2, and triggered calcium release. RT-PCR examination indicated that IsoF up-regulated tumor necrosis factor (TNF)α, angiopoietin-1 receptor (Tie2), and vascular endothelial growth factor (VEGF)-A, but did not interfere with caspase 2 and VEGF-C in the RBECs. IsoF has pro-angiogenic activity. Calcium release and ERK1/2 phosphorylation may be involved in the signaling of the IsoF-induced up-regulation of TNFα, Tie2, and VEGF-A, which could be the molecular mechanism of the pro-angiogenic activity of the IsoF.

Angiopoietin-1 Is Required for Vortex Vein and Choriocapillaris Development in Mice.

BACKGROUND: The choroidal vasculature, including the choriocapillaris and vortex veins, is essential for providing nutrients to the metabolically demanding photoreceptors and retinal pigment epithelium. Choroidal vascular dysfunction leads to vision loss and is associated with age-related macular degeneration and the poorly understood pachychoroid diseases including central serous chorioretinopathy and polypoidal choroidal vasculopathy that are characterized by formation of dilated pachyvessels throughout the choroid. METHODS: Using neural crest-specific Angpt1 knockout mice, we show that Angiopoietin 1, a ligand of the endothelial receptor TEK (also known as Tie2) is essential for choriocapillaris development and vortex vein patterning. RESULTS: Lacking choroidal ANGPT1, neural crest-specific Angpt1 knockout eyes exhibited marked choriocapillaris attenuation and 50% reduction in number of vortex veins, with only 2 vortex veins present in the majority of eyes. Shortly after birth, dilated choroidal vessels resembling human pachyvessels were observed extending from the remaining vortex veins and displacing the choriocapillaris, leading to retinal pigment epithelium dysfunction and subretinal neovascularization similar to that seen in pachychoroid disease. CONCLUSIONS: Together, these findings identify a new role for ANGPT1 in ocular vascular development and demonstrate a clear link between vortex vein dysfunction, pachyvessel formation, and disease.

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.