Ang1/Tie2/VE-Cadherin Signaling Regulates DPSCs in Vascular Maturation.

Adding dental pulp stem cells (DPSCs) to vascular endothelial cell-formed vessel-like structures can increase the longevity of these vessel networks. DPSCs display pericyte-like cell functions and closely assemble endothelial cells (ECs). However, the mechanisms of DPSC-derived pericyte-like cells in stabilizing the vessel networks are not fully understood. In this study, we investigated the functions of E-DPSCs, which were DPSCs isolated from the direct coculture of human umbilical vein endothelial cells (HUVECs) and DPSCs, and T-DPSCs, which were DPSCs treated by transforming growth factor beta 1 (TGF-ß1), in stabilizing blood vessels in vitro and in vivo. A 3-dimensional coculture spheroid sprouting assay was conducted to compare the functions of E-DPSCs and T-DPSCs in vitro. Dental pulp angiogenesis in the severe combined immunodeficiency (SCID) mouse model was used to explore the roles of E-DPSCs and T-DPSCs in vascularization in vivo. The results demonstrated that both E-DPSCs and T-DPSCs possess smooth muscle cell-like cell properties, exhibiting higher expression of the mural cell-specific markers and the suppression of HUVEC sprouting. E-DPSCs and T-DPSCs inhibited HUVEC sprouting by activating TEK tyrosine kinase (Tie2) signaling, upregulating vascular endothelial (VE)-cadherin, and downregulating vascular endothelial growth factor receptor 2 (VEGFR2). In vivo study revealed more perfused and total blood vessels in the HUVEC + E-DPSC group, HUVEC + T-DPSC group, angiopoietin 1 (Ang1) pretreated group, and vascular endothelial protein tyrosine phosphatase (VE-PTP) inhibitor pretreated group, compared to HUVEC + DPSC group. In conclusion, these data indicated that E-DPSCs and T-DPSCs could stabilize the newly formed blood vessels and accelerate their perfusion. The critical regulating pathways are Ang1/Tie2/VE-cadherin and VEGF/VEGFR2 signaling.

Anti-Arthritic Effect of Edaravone Against Complete Freund Adjuvant Induced Arthritis via Osteoclast Differentiation and HIF-1α-VEGF-ANG-1 Axis.

Background: Bone dysfunction is a crucial problem that occurs during rheumatoid arthritis (RA) disease. Osteoclast plays a significant role in bone resorption and osteoclast differentiation and its enhancement of bone destruction. Edaravone remarkably exhibited free radical scavenging and anti-inflammatory effects. The objective of the current investigation is to comfort the inhibitory effect of Edaravone (ED) against complete Freund adjuvant (CFA) rat model via inhibition of angiogenesis and inflammation. Methods: Subcutaneous injection of CFA (1%) was used to induce arthritis; the rats were divided into different groups and received the oral administration of ED. Paw edema, body weight, and arthritis score were regularly estimated. Biochemical parameters were estimated, respectively. We also estimate the level of hypoxia-inducible factor-1α (HIF-1α), angiopoietin 1 (ANG-1), and vascular endothelial growth factor (VEGF). We also checked into how ED affected the differentiation of osteoclasts utilising a co-culture system with monocytes and synovial fibroblasts in arthritis rats. Results: ED treatment significantly (P<0.001) suppressed the arthritis score and paw edema and improved the body weight. ED treatment significantly (P<0.001) altered the antioxidant parameters and pro-inflammatory cytokines: inflammatory mediator nuclear kappa B factor (NF-κB), cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2), respectively. Furthermore, ED treatment significantly (P<0.001) suppressed the level of ANG-1, HIF-1α, and VEGF, respectively. The results suggest that ED suppressed osteoclast differentiation and also decreased the level of cytokines and osteopontin (OPN), receptor activator for nuclear factor-κ B Ligand (RANKL) and macrophage colony stimulating factor (M-CSF) in the co-culture supernatant of monocytes and synovial fibroblasts. Conclusion: Edaravone could mitigate CFA via inhibiting angiogenesis and inflammatory reactions, which may be linked with the HIF-1α-VEGF-ANG-1 axis and also enhance the bone destruction of murine arthritis via suppression of osteoclast differentiation and inflammatory reaction.

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.

Ang-1 Inhibited Endoplasmic Reticulum Stress and Apoptosis of VECs in Rats with aSAH-induced CVS Through the Regulated PI3K/Akt Pathway.

AIMS: To explore angiopoietin-1 (Ang-1) involved in cerebral vasospasm (CVS) after aneurysmal subarachnoid hemorrhage (aSAH) through its effect on endoplasmic reticulum stress (ERS) and apoptosis of vascular endothelial cells (VECs). BACKGROUND: CVS accounts for high morbidity and mortality of aSAH. Abnormal cellular physiological processes of VECs play a critical role in aSAH-induced CVS. In addition, Ang-1 is involved in regulating vascular structure and function. OBJECTIVE: To study the role of Ang-1 played in CVS and the underlying mechanism. METHODS: Blood samples of 130 aSAH patients were collected from 2016 to 2020 at West China Hospital of Sichuan University. A two-hemorrhage rodent model was employed to structure an aSAH-induced CVS rat model. Moreover, oxyHb was used to treat VECs to construct a CVS cell model in vitro. ELISA was used to measure the level of Ang-1 and HE staining to assess the rat's basilar arteries. Subsequently, CCK-8 was used to detect cell viability ability, and flow cytometry was used to test the cell apoptosis rate. Western blotting was used to determine the expression level of ERS marker and apoptosis-related proteins. RESULTS: There was an abnormally low expression of Ang-1 in CVS patients and CVS rats; besides, oxyHb treatment decreased Ang-1 in VECs in a concentration-dependent manner. Ang-1 treatment led to the thinner basilar artery wall and lumen circumference in CVS rats; moreover, in oxyHbtreated VECs, Ang-1 treatment inhibited ERS and apoptosis. In addition, the expression of p-PI3K and p-Akt in the CVS group decreased, while the expression of p53 in the CVS group increased. The expression of p-PI3K and p-Akt in 8 CVS rats negatively correlates with the expression of Ang- 1, but the correlation between p53 and Ang-1 was positive. Furthermore, the results suggested that Ang-1 suppressed ERS and apoptosis of VECs through the regulated PI3K/Akt/p53 pathway. CONCLUSION: Elevated Ang-1 inhibited p53-mediated ERS and apoptosis of VECs through the activated PI3K/Akt pathway; Ang-1 might be an attractive treatment strategy for CVS.

Combined Effects of Polydopamine-Assisted Copper Immobilization on 3D-Printed Porous Ti6Al4V Scaffold for Angiogenic and Osteogenic Bone Regeneration.

Numerous studies have demonstrated that biological compounds and trace elements such as dopamine (DA) and copper ions (Cu) could be modified onto the surfaces of scaffolds using a one-step immersion process which is simple, inexpensive and, most importantly, non-cytotoxic. The development and emergence of 3D printing technologies such as selective laser melting (SLM) have also made it possible for us to fabricate bone scaffolds with precise structural designs using metallic compounds. In this study, we fabricated porous titanium scaffolds (Ti) using SLM and modified the surface of Ti with polydopamine (PDA) and Cu. There are currently no other reported studies with such a combination for osteogenic and angiogenic-related applications. Results showed that such modifications did not affect general appearances and microstructural characteristics of the porous Ti scaffolds. This one-step immersion modification allowed us to modify the surfaces of Ti with different concentrations of Cu ions, thus allowing us to fabricate individualized scaffolds for different clinical scenarios. The modification improved the hydrophilicity and surface roughness of the scaffolds, which in turn led to promote cell behaviors of Wharton's jelly mesenchymal stem cells. Ti itself has high mechanical strength, therefore making it suitable for surgical handling and clinical applications. Furthermore, the scaffolds were able to release ions in a sustained manner which led to an upregulation of osteogenic-related proteins (bone alkaline phosphatase, bone sialoprotein and osteocalcin) and angiogenic-related proteins (vascular endothelial growth factor and angiopoietin-1). By combining additive manufacturing, Ti6Al4V scaffolds, surface modification and Cu ions, the novel hybrid 3D-printed porous scaffold could be fabricated with ease and specifically benefited future bone regeneration in the clinic.

Secretome from Human Mesenchymal Stem Cells-Derived Endothelial Cells Promotes Wound Healing in a Type-2 Diabetes Mouse Model.

Tissue regeneration is often impaired in patients with metabolic disorders such as diabetes mellitus and obesity, exhibiting reduced wound repair and limited regeneration capacity. We and others have demonstrated that wound healing under normal metabolic conditions is potentiated by the secretome of human endothelial cell-differentiated mesenchymal stem cells (hMSC-EC). However, it is unknown whether this effect is sustained under hyperglycemic conditions. In this study, the wound healing effect of secretomes from undifferentiated human mesenchymal stem cells (hMSC) and hMSC-EC in a type-2 diabetes mouse model was analyzed. hMSC were isolated from human Wharton's jelly and differentiated into hMSC-EC. hMSC and hMSC-EC secretomes were analyzed and their wound healing capacity in C57Bl/6J mice fed with control (CD) or high fat diet (HFD) was evaluated. Our results showed that hMSC-EC secretome enhanced endothelial cell proliferation and wound healing in vivo when compared with hMSC secretome. Five soluble proteins (angiopoietin-1, angiopoietin-2, Factor de crecimiento fibroblástico, Matrix metallopeptidase 9, and Vascular Endothelial Growth Factor) were enriched in hMSC-EC secretome in comparison to hMSC secretome. Thus, the five recombinant proteins were mixed, and their pro-healing property was evaluated in vitro and in vivo. Functional analysis demonstrated that a cocktail of these proteins enhanced the wound healing process similar to hMSC-EC secretome in HFD mice. Overall, our results show that hMSC-EC secretome or a combination of specific proteins enriched in the hMSC-EC secretome enhanced wound healing process under hyperglycemic conditions.

Angiopoietin-1 protects against endotoxin-induced neonatal lung injury and alveolar simplification in mice.

BACKGROUND: Sepsis in premature newborns is a risk factor for bronchopulmonary dysplasia (BPD), but underlying mechanisms of lung injury remain unclear. Aberrant expression of endothelial cell (EC) angiopoietin 2 (ANGPT2) disrupts angiopoietin 1 (ANGPT1)/TIE2-mediated endothelial quiescence, and is implicated in sepsis-induced acute respiratory distress syndrome in adults. We hypothesized that recombinant ANGPT1 will mitigate sepsis-induced ANGPT2 expression, inflammation, acute lung injury (ALI), and alveolar remodeling in the saccular lung. METHODS: Effects of recombinant ANGPT1 on lipopolysaccharide (LPS)-induced endothelial inflammation were evaluated in human pulmonary microvascular endothelial cells (HPMEC). ALI and long-term alveolar remodeling were assessed in newborn mice exposed to intraperitoneal LPS and recombinant ANGPT1 pretreatment. RESULTS: LPS dephosphorylated EC TIE2 in association with increased ANGPT2 in vivo and in vitro. ANGPT1 suppressed LPS and ANGPT2-induced EC inflammation in HPMEC. Neonatal mice treated with LPS had increased lung cytokine expression, neutrophilic influx, and cellular apoptosis. ANGPT1 pre-treatment suppressed LPS-induced lung Toll-like receptor signaling, inflammation, and ALI. LPS-induced acute increases in metalloproteinase 9 expression and elastic fiber breaks, as well as a long-term decrease in radial alveolar counts, were mitigated by ANGPT1. CONCLUSIONS: In an experimental model of sepsis-induced BPD, ANGPT1 preserved endothelial quiescence, inhibited ALI, and suppressed alveolar simplification. IMPACT: Key message: Angiopoietin 1 inhibits LPS-induced neonatal lung injury and alveolar remodeling. Additions to existing literature: Demonstrates dysregulation of angiopoietin-TIE2 axis is important for sepsis- induced acute lung injury and alveolar simplification in experimental BPD. Establishes recombinant Angiopoietin 1 as an anti-inflammatory therapy in BPD. IMPACT: Angiopoietin 1-based interventions may represent novel therapies for mitigating sepsis-induced lung injury and BPD in premature infants.

Combined treatment with C16 peptide and angiopoietin-1 confers neuroprotection and reduces inflammation in 3-nitropropionic acid-induced dystonia mice.

Dystonia is a disorder associated with abnormalities in many brain regions including the basal ganglia and cerebellum. The toxin 3-Nitropropionic acid (3-NP) can induce neuropathologies in the mice striatum and nigra substance, including excitotoxicity, neuroinflammation, and extensive neuronal atrophy, characterized by progressive motor dysfunction, dystonia, and memory loss, mimicking those observed in humans. We established a mouse model of dystonia by administering 3-NP. Given the reported neuroprotective effects of the endothelial growth factor angiopoietin-1 (Ang-1) and the anti-inflammatory integrin αvß3 binding peptide C16, we performed this study to evaluate their combined effects on 3-NP striatal toxicity and their therapeutic potential with multiple methods using an in vivo mouse model. Sixty mice were equally and randomly divided into three groups: control, 3-NP treatment, and 3-NP+C16+Ang-1 treatment. Behavioral and electrophysiological tests were conducted and the effect of the combined C16+Ang-1 treatment on neural function recovery was determined. We found that C16+Ang-1 treatment alleviated 3-NP-induced behavioral, biochemical, and cellular alterations in the central nervous system and promoted function recovery by restoring vascular permeability and reducing inflammation in the micro-environment. In conclusion, our results confirmed the neuroprotective effect of combined C16+Ang-1 treatment and suggest their potential as a complementary therapeutic against 3-NP-induced dystonia.

Angiopoietins stimulate pancreatic islet development from stem cells.

In vitro differentiation of human induced pluripotent stem cells (iPSCs) into functional islets holds immense potential to create an unlimited source of islets for diabetes research and treatment. A continuous challenge in this field is to generate glucose-responsive mature islets. We herein report a previously undiscovered angiopoietin signal for in vitro islet development. We revealed, for the first time, that angiopoietins, including angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) permit the generation of islets from iPSCs with elevated glucose responsiveness, a hallmark of mature islets. Angiopoietin-stimulated islets exhibited glucose synchronized calcium ion influx in repetitive glucose challenges. Moreover, Ang2 augmented the expression of all islet hormones, including insulin, glucagon, somatostatin, and pancreatic polypeptide; and ß cell transcription factors, including NKX6.1, MAFA, UCN3, and PDX1. Furthermore, we showed that the Ang2 stimulated islets were able to regulate insulin exocytosis through actin-filament polymerization and depolymerization upon glucose challenge, presumably through the CDC42-RAC1-gelsolin mediated insulin secretion signaling pathway. We also discovered the formation of endothelium within the islets under Ang2 stimulation. These results strongly suggest that angiopoietin acts as a signaling molecule to endorse in vitro islet development from iPSCs.

The protective effects of C16 peptide and angiopoietin-1 compound in lipopolysaccharide-induced acute respiratory distress syndrome.

C16 peptide and angiopoietin-1 (Ang-1) have been found to have anti-inflammatory activity in various inflammation-related diseases. However, their combined role in acute respiratory distress syndrome (ARDS) has not been investigated yet. The objective of this study was to investigate the effects of C16 peptide and Ang-1 in combination with lipopolysaccharide (LPS)-induced inflammatory insult in vitro and in vivo. Human pulmonary microvascular endothelial cells and human pulmonary alveolar epithelial cells were used as cell culture systems, and an ARDS rodent model was used for in vivo studies. Our results demonstrated that C16 and Ang-1 in combination significantly suppressed inflammatory cell transmigration by 33% in comparison with the vehicle alone, and decreased the lung tissue wet-to-dry lung weight ratio to a maximum of 1.53, compared to 3.55 in the vehicle group in ARDS rats. Moreover, C + A treatment reduced the histology injury score to 60% of the vehicle control, enhanced arterial oxygen saturation (SO2), decreased arterial carbon dioxide partial pressure (PCO2), and increased oxygen partial pressure (PO2) in ARDS rats, while also improving the survival rate from 47% (7/15) to 80% (12/15) and diminishing fibrosis, necrosis, and apoptosis in lung tissue. Furthermore, when C + A therapy was administered 4 h following LPS injection, the treatment showed significant alleviating effects on pulmonary inflammatory cell infiltration 24 h postinsult. In conclusion, our in vitro and in vivo studies show that C16 and Ang-1 exert protective effects against LPS-induced inflammatory insult. C16 and Ang-1 hold promise as a novel agent against LPS-induced ARDS. Further studies are needed to determine the potential for C16 and Ang-1 in combination in treating inflammatory lung diseases.

C16 peptide and angiopoietin-1 protect against LPS-induced BV-2 microglial cell inflammation.

AIMS: Pathological alterations in the brain can cause microglial activation (MA). Thus, inhibiting MA could provide a new approach for treating neurodegenerative disorders. MAIN METHODS: To investigate the effect of C16 peptide and angiopoietin-1 (Ang1) on inflammation following MA, we stimulated microglial BV-2 cells with lipopolysaccharide (LPS) and used dexmedetomidine (DEX) as a positive control. Specific inhibitors of Tie2, αvß3 and α5ß1 integrins, and PI3K/Akt were applied to investigate the neuron-protective and anti-inflammatory effects and signaling pathway of C16 + Ang1 treatment in the LPS-induced BV-2 cells. KEY FINDINGS: Our results showed that C16 + Ang1 treatment reduced the microglia M1 phenotype but promoted the microglia M2 phenotype. In addition, C16 + Ang1 treatment suppressed leukocyte migration across human pulmonary microvascular endothelial cells, reduced the levels of pro-inflammatory factors [inducible nitric oxide synthase (iNOS), interleukin (IL)-1ß, tumor necrosis factor (TNF-α)], and cellular apoptosis factors (caspase-3 and p53), and decreased lactate dehydrogenase (LDH) release, but promoted anti-inflammatory cytokine (IL-10) expression and cell proliferation in the LPS-activated BV-2 cells. The signaling pathways underlying the neuron-protective and anti-inflammatory effects of C16 + Ang1 may be mediated by Tie2-PI3K/Akt, Tie2-integrin and integrin-PI3K/Akt. SIGNIFICANCE: The neuron-protective and anti-inflammatory effects of C16 + Ang1 treatment included M1 to M2 microglia phenotype switching, blocking leukocyte transmigration, decreasing apoptotic and inflammatory factors, and promoting cellular viability.

Angiopoietin-1 protects 3T3-L1 preadipocytes from saturated fatty acid-induced cell death.

Cross talk between endothelial cells and adipocytes is vital to adipocyte functions, but little is known about the mechanisms or factors controlling the process. Angiogenesis is a critical component linking the endothelium to healthy adipogenesis, yet it is not known if or how it is involved in adipocyte physiology. Therefore, the purpose of this study was to determine the effect of angiopoietin-1 (Ang-1) and -2 (Ang-2) as well as their receptor, Tie-2, on adipocyte physiology. 3T3-L1 pre- and mature adipocytes were found to express Ang-1, Ang-2, and Tie-2, which decrease upon polyunsaturated fatty acid treatment. Furthermore, 3T3-L1 cells treated with recombinant Ang-1 or Ang-2 increased expression of the antiapoptotic gene Bcl-x and decreased expression of the proapoptotic gene Casp-8. Next, preadipocytes were treated with saturated fatty acids (SFAs) to induce cell stress. SFA-mediated splicing of X-box-binding protein-1 was reduced by co-treatment with Ang-1, and cell viability was improved in the presence of SFAs + Ang-1. Taken together, these results indicate that Ang-1 may protect preadipocytes from SFA-induced apoptosis and endoplasmic reticulum stress.

NOX2, NOX4, and mitochondrial-derived reactive oxygen species contribute to angiopoietin-1 signaling and angiogenic responses in endothelial cells.

Angiopoietin-1 (Ang-1) is a ligand of Tie-2 receptors that promotes survival, migration, and differentiation of endothelial cells. Several studies have linked reactive oxygen species (ROS) to Ang-1 signaling and distinct angiogenic responses, but the molecular sources of these ROS have never been clearly identified. In this study, we have identified source-specific contributions of ROS to Ang-1/Tie 2 signaling and angiogenic responses in human umbilical vein endothelial cells (HUVECs), specifically the differential contributions of mitochondrial ROS (mtROS) and ROS from two isoforms of NADPH oxidase (NOX2, NOX4). We demonstrate that: 1) Ang-1 induces significant increases in mtROS production under normal conditions but does not when cells are pre-incubated with mitochondrial antioxidants; 2) Ang-1 induces rapid Tie-2-dependent increases in cytosolic ROS production but does not when NOX2 and NOX4 are knocked down; 3) Ang-1 induces simultaneous increases in phosphorylation of AKT, ERK1/2, p38, and SAPK/JNK proteins within a few minutes of exposure, but this response is strongly and selectively attenuated when NOX2 and NOX4 are knocked down or cells are pre-treated with mitochondrial antioxidants; 4) Ang-1 exerts a strong effect on HUVEC survival in serum-deprived medium and enhances cell migration and capillary tube formation, but the survival response is inhibited by NOX2 knockdown and the migration and tube formation responses are entirely absent with NOX4 knockdown or pre-treatment with mitochondrial antioxidants. We conclude that Ang-1 triggers NOX2, NOX4, and the mitochondria to release ROS and that ROS derived from these sources play distinct roles in the regulation of the Ang-1/Tie 2 signaling pathway and pro-angiogenic responses.

Modulation of long-term endothelial-barrier integrity is conditional to the cross-talk between Akt and Src signaling.

Although numerous studies have implicated Akt and Src kinases in vascular endothelial growth factor (VEGF) and Angiopoietin-1 (Ang-1)-induced endothelial-barrier regulation, a link between these two pathways has never been demonstrated. We determined the long-term effects of Akt inhibition on Src activity and vice versa, and in turn, on the human microvascular endothelial cell (HMEC) barrier integrity at the basal level, and in response to growth factors. Our data showed that Akt1 gene knockdown increases gap formation in HMEC monolayer at the basal level. Pharmacological inhibition of Akt, but not Src resulted in exacerbated VEGF-induced vascular leakage and impaired Ang-1-induced HMEC-barrier protection in vitro at 24 hr. Whereas inhibition of Akt had no effect on VEGF-induced HMEC gap formation in the short term, inhibition of Src blunted this process. In contrast, inhibition of Akt disrupted the VEGF and Ang-1 stabilized barrier integrity in the long-term while inhibition of Src did not. Interestingly, both long-term Akt inhibition and Akt1 gene knockdown in HMECs resulted in increased Tyr416 phosphorylation of Src. Treatment of HMECs with transforming growth factor-ß1 (TGFß1) that inhibited Akt Ser473 phosphorylation in the long-term, activated Src through increased Tyr416 phosphorylation and decreased HMEC-barrier resistance. The effect of TGFß1 on endothelial-barrier breakdown was blunted in Akt1 deficient HMEC monolayers, where endothelial-barrier resistance was already impaired compared to the control. To our knowledge, this is the first report demonstrating a direct cross-talk between Akt and Src in endothelial-barrier regulation.

Molecular Changes Associated with the Protective Effects of Angiopoietin-1 During Blood-Brain Barrier Breakdown Post-Injury.

Blood-brain barrier (BBB) breakdown to plasma proteins leads to vasogenic edema which when diffuse is a life threatening complication in many types of acute brain injury. In our previous studies, early BBB breakdown was associated with increased expression of endothelial caveolin-1α (Cav-1) protein and decreased expression of occludin. In order to attenuate these changes, the effects of intra-cortical angiopoietin-1 (Ang1), a potent anti-permeability factor, on BBB breakdown was assessed in the cold injury model at day 1 post-injury. Overall vascular permeability at the lesion site was assessed in Ang1 non-treated and treated cold-injured rats, using horseradish peroxidase (HRP) as a tracer and in individual vessels by dual labeling immunofluorescence for Cav-1 or occludin and fibronectin, a marker of BBB breakdown. In addition, Cav-1, occludin, Akt, and ERK1/2 expression at the lesion site was detected by immunoblotting. Non-treated cold-injured rats showed focal HRP leakage at the lesion site which was significantly decreased (P < 0.001) in the Ang1-treated group. Increased endothelial Cav-1 and decreased occludin immunoreactivity was observed in arterioles and corresponding-sized venules with BBB breakdown in the non-treated cold-injured rats, and similar expression of these proteins was detected at the lesion site by immunoblotting associated with increased expression of Akt and ERK2 proteins. These alterations were attenuated by Ang1 treatment which resulted in Cav-1, occludin, Akt, and ERK1/2 protein expression that was similar to that of the control groups as was the endothelial Cav-1 and occludin immunoreactivity in leaky vessels. These data suggest that Ang1 administered early post-injury has potential in attenuating the degree of vascular alterations and subsequent vasogenic edema.

Dual delivery of VEGF and ANG-1 in ischemic hearts using an injectable hydrogel.

Acute myocardial infarction (MI) caused by ischemia is the most common cause of cardiac dysfunction. While growth factor therapy is promising, the retention in the highly vascularized myocardium is limited and prevents sustained activation needed for adequate cellular responses. Here, we demonstrated the use of polyethylene glycol-fibrinogen (PF) hydrogels for sustained dual delivery of vascular endothelial growth factor (VEGF) and angiopoietin-1 (ANG-1) to enhance myocardial repair and function. VEGF and ANG-1 were incorporated in PF hydrogels and their in vitro characteristics were studied. Acute MI was generated in a rodent model with rats randomly assigned to 4 groups; sham, saline, PF and PF-VEGF-ANG1 (n=10 each group). Saline or hydrogel was injected in infarct and peri-infarct areas of the myocardium. After 4weeks, myocardial function was assessed using echocardiography. Tissue samples were harvested for Hematoxylin and Eosin, Masson Trichrome and capillary staining to assess the extent of fibrotic scar and arteriogenesis. Both VEGF and ANG-1 were released in a sustained and controlled manner over 30days. PF-VEGF-ANG1 treated animals showed the best improvement in cardiac function, highest degree of cardiac muscle preservation, and arteriogenesis. This study demonstrates that PF hydrogels can simultaneously provide mechanical support to attenuate adverse myocardial remodelling, and a pro-angiogenic benefit from the sustained VEGF and ANG1 delivery that culminates in a restorative effect following MI. The utility of this synergistic, biomaterial-based growth factor delivery may have clinical implications in the prevention of post-MI cardiac dysfunction. STATEMENT OF SIGNIFICANCE: Acute myocardial infarction (MI) caused by ischemia is the most common cause of cardiac dysfunction. Here, we demonstrated the use of polyethylene glycol-fibrinogen (PF) hydrogels for sustained dual delivery of vascular endothelial growth factor (VEGF) and angiopoietin-1 (ANG-1) to enhance myocardial repair and function. Treated animals showed the best improvement in cardiac function, highest degree of cardiac muscle preservation, and arteriogenesis. This study demonstrates that PF hydrogels can simultaneously provide mechanical support to attenuate adverse myocardial remodelling, and a pro-angiogenic benefit from the sustained VEGF and ANG1 delivery that culminates in a restorative effect following MI.

Aflibercept and Ang1 supplementation improve neoadjuvant or adjuvant chemotherapy in a preclinical model of resectable breast cancer.

Phase III clinical trials evaluating bevacizumab (an antibody to the angiogenic ligand, VEGF-A) in breast cancer have found improved responses in the presurgical neoadjuvant setting but no benefits in the postsurgical adjuvant setting. The objective of this study was to evaluate alternative antiangiogenic therapies, which target multiple VEGF family members or differentially modulate the Angiopoietin/Tie2 pathway, in a mouse model of resectable triple-negative breast cancer (TNBC). Neoadjuvant therapy experiments involved treating established orthotopic xenografts of an aggressive metastatic variant of the MDA-MB-231 human TNBC cell line, LM2-4. Adjuvant therapies were given after primary tumor resections to treat postsurgical regrowths and distant metastases. Aflibercept ('VEGF Trap', which neutralizes VEGF-A, VEGF-B and PlGF) showed greater efficacy than nesvacumab (an anti-Ang2 antibody) as an add-on to neoadjuvant/adjuvant chemotherapy. Concurrent inhibition of Ang1 and Ang2 signaling (through an antagonistic anti-Tie2 antibody) was not more efficacious than selective Ang2 inhibition. In contrast, short-term perioperative BowAng1 (a recombinant Ang1 variant) improved the efficacy of adjuvant chemotherapy. In conclusion, concurrent VEGF pathway inhibition is more likely than Ang/Tie2 pathway inhibition (e.g., anti-Ang2, anti-Ang2/Ang1, anti-Tie2) to improve neoadjuvant/adjuvant chemotherapies for TNBC. Short-term perioperative Ang1 supplementation may also have therapeutic potential in conjunction with adjuvant chemotherapy for TNBC.

Fibulin-5 Regulates Angiopoietin-1/Tie-2 Receptor Signaling in Endothelial Cells.

BACKGROUND: Fibulin-5 is an extracellular matrix glycoprotein that plays critical roles in vasculogenesis and embryonic development. Deletion of Fibulin-5 in mice results in enhanced skin vascularization and upregulation of the angiogenesis factor angiopoietin-1 (Ang-1), suggesting that Fibulin-5 functions as an angiogenesis inhibitor. In this study, we investigate the inhibitory effects of Fibulin-5 on Ang-1/TIE-2 receptor pathway signaling and cell survival in human endothelial cells. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant wild-type and RGE-mutant Fibulin-5 proteins were generated through stable transfection of HEK293 and CHO cells, respectively. In vitro solid phase binding assays using pure proteins revealed that wild-type Fibulin-5 does not bind to Ang-1 or TIE-2 proteins but strongly binds to heparin. Binding assays using human umbilical vein endothelial cells (HUVECs) indicated that wild-type Fibulin-5 strongly binds to cells but RGE-mutant Fibulin-5, which is incapable of binding to integrins, does not. Pre-incubation of HUVECs for 1 hr with Fibulin-5 significantly increased caspase 3/7 activity, ERK1/2 phosphorylation, and expressions of the transcription factor early growth response 1 (EGR1) and the dual-specificity phosphatase 5 (DUSP5). Fibulin-5 also strongly attenuated Ang-1-induced TIE-2 and AKT phosphorylation, decreased Ang-1-induced expressions of the transcription factors Inhibitor of DNA Binding 1 (ID1) and Kruppel-like Factor 2 (KLF2), and reversed the inhibitory effect of Ang-1 on serum deprivation-induced cytotoxicity and caspase 3/7 activity. CONCLUSION/SIGNIFICANCE: We conclude that Fibulin-5 strongly binds to the endothelial cell surface through heparin-sulfate proteoglycans and possibly integrins and that it exerts strong anti-angiogenic effects by reducing endothelial cell viability and interfering with the signaling pathways of the Ang-1/TIE-2 receptor axis.

Rational Design of Prevascularized Large 3D Tissue Constructs Using Computational Simulations and Biofabrication of Geometrically Controlled Microvessels.

A major challenge in the development of clinically relevant 3D tissue constructs is the formation of vascular networks for oxygenation, nutrient supply, and waste removal. To this end, this study implements a multimodal approach for the promotion of vessel-like structures formation in stiff fibrin hydrogels. Computational simulations have been performed to identify the easiest microchanneled configuration assuring normoxic conditions throughout thick cylindrical hydrogels (8 mm height, 6 mm ∅), showing that in our configuration a minimum of three microchannels (600 µm ∅), placed in a non-planar disposition, is required. Using small hydrogel bricks with oxygen distribution equal to the microchanneled configuration, this study demonstrates that among different culture conditions, co-culture of mesenchymal and endothelial cells supplemented with ANG-1 and VEGF leads to the most developed vascular network. Microchanneled hydrogels have been then cultured in the same conditions both statically and in a bioreactor for 7 d. Unexpectedly, the combination between shear forces and normoxic conditions is unable to promote microvascular networks formation in three-channeled hydrogels. Differently, application of either shear forces or normoxic conditions alone results in microvessels outgrowth. These results suggest that to induce angiogenesis in engineered constructs, complex interactions between several biochemical and biophysical parameters have to be modulated.

Akt1 promotes stimuli-induced endothelial-barrier protection through FoxO-mediated tight-junction protein turnover.

Vascular permeability regulated by the vascular endothelial growth factor (VEGF) through endothelial-barrier junctions is essential for inflammation. Mechanisms regulating vascular permeability remain elusive. Although 'Akt' and 'Src' have been implicated in the endothelial-barrier regulation, it is puzzling how both agents that protect and disrupt the endothelial-barrier activate these kinases to reciprocally regulate vascular permeability. To delineate the role of Akt1 in endothelial-barrier regulation, we created endothelial-specific, tamoxifen-inducible Akt1 knockout mice and stable ShRNA-mediated Akt1 knockdown in human microvascular endothelial cells. Akt1 loss leads to decreased basal and angiopoietin1-induced endothelial-barrier resistance, and enhanced VEGF-induced endothelial-barrier breakdown. Endothelial Akt1 deficiency resulted in enhanced VEGF-induced vascular leakage in mice ears, which was rescued upon re-expression with Adeno-myrAkt1. Furthermore, co-treatment with angiopoietin1 reversed VEGF-induced vascular leakage in an Akt1-dependent manner. Mechanistically, our study revealed that while VEGF-induced short-term vascular permeability is independent of Akt1, its recovery is reliant on Akt1 and FoxO-mediated claudin expression. Pharmacological inhibition of FoxO transcription factors rescued the defective endothelial barrier due to Akt1 deficiency. Here we provide novel insights on the endothelial-barrier protective role of VEGF in the long term and the importance of Akt1-FoxO signaling on tight-junction stabilization and prevention of vascular leakage through claudin expression.