VEGF-induced Endothelial Cell Research Articles

Inhibitory Effect of Phenolic Compounds on Vascular Endothelial Growth Factor-Induced Retinal Endothelial Permeability and Angiogenesis.

Age-related macular degeneration (AMD), often triggered by endothelial barrier disruption through vascular endothelial growth factor (VEGF), is a leading cause of blindness. This study investigated the inhibitory effects of phenolic compounds on VEGF-induced endothelial cell proliferation, migration, angiogenesis, and permeability using human retinal microvascular endothelial cells (hRECs). Thirty-seven polyphenolic compounds were selected from various databases based on their antioxidant properties, abundance in food, and solubility. These compounds significantly reduced migration, tube formation, and endothelial permeability in VEGF-stimulated hRECs. Notably, formononetin, eriodictyol, biochanin A, and p-coumaric acid were more effective in suppressing VEGF-induced angiogenesis and endothelial permeability than lutein. Molecular docking simulations revealed that formononetin, eriodictyol, and biochanin A had relatively lower binding energies with VEGF receptor 2 (VEGFR2) than lutein and sorafenib. These findings highlight the potential of phenolic compounds to be used as VEGFR2 inhibitors and an alternative strategy for preventing AMD.

Regulation of angiogenesis by signal sequence-derived peptides.

The neuropilin-like, Discoidin, CUB and LCCL domain containing 2 (DCBLD2) is a transmembrane protein with an unusually long signal sequence (SS) composed of N-terminal (N) and C-terminal (C) subdomains, separated by a transition (tra) subdomain. DCBLD2 interacts with VEGFR-2 and regulates VEGF-induced endothelial cell signaling, proliferation and migration, as well as angiogenesis. The exact mechanisms by which DCBLD2 interacts with VEGFR2 to modulate VEGF signaling remain unclear. Searching for VEGFR2 interacting DCBLD2 domains, we generated various constructs containing different DCBLD2 domain combinations and conducted co-immunoprecipitation and signaling studies in HEK 293T and endothelial cells. Several peptides were synthesized based on the identified domain, and their effect on VEGF signaling was assessed in vitro in cell culture and in vivo using matrigel plug and corneal micropocket assays. The effect of the lead peptide was further evaluated using a murine hindlimb ischemia model. DCBLD2 SS interacted with VEGFR2 and promoted VEGF signaling. SS was not cleaved in the mature DCBLD2 and its hydrophobic transmembrane 'traC' segment, but not the 'N' subdomain, was involved in DCBLD2-VEGFR2 interaction. The smallest unit in DCBLD2 SS that interacts with VEGFR2 was the L5VL5 sequence. Even after the central valine was removed, the L10 sequence mimicked the DCBLD2 SS traC's effect on VEGF-signaling, while shorter or longer poly-leucine sequences were less effective. Finally, a synthetic traC peptide enhanced VEGF signaling in vitro, promoted VEGF-induced angiogenesis in vivo, and improved blood flow recovery following hindlimb ischemia. DCBLD2 SS along with its derivative peptides can promote VEGFR2 signaling and angiogenesis. Synthetic peptides based on DCBLD2 SS hold promise as therapeutic agents for regulating angiogenesis. Importantly these findings refine the traditional view of signal sequences as mere targeting elements, revealing a role in cellular signaling. This opens new avenues for research and therapeutic strategies.

Abstract 5385: Synthetic disulfide derivative of vitamin B1, fursultiamine, prevents VEGF-induced angiogenesis

Abstract Angiogenesis is a process in which new blood vessels are formed from the existing blood vessels required to supply nutrients and oxygen to various tissues. Angiogenesis plays a significant role in the growth and spread of many cancers, and several anti-angiogenic agents act as chemotherapeutic agents. Recent studies have shown that fursultiamine (thiamine tetrahydrofurfuryl disulfide), a lipid-soluble synthetic disulfide derivative of thiamine, possesses viable antineoplastic and antiviral effects. However, its role in preventing angiogenesis is not known. We hypothesize that fursultiamine, with its potent antioxidative actions, prevents VEGF-induced angiogenesis in vitro and in vivo. Human umbilical vein endothelial cells (HUVECs) were treated with VEGF in the absence and presence of fursultiamine (0-100 uM) in a time and dose-dependent manner. Cell viability was determined by MTT assay and in vitro angiogenesis by tube formation assays. The expression of various pro-angiogenic, anti-angiogenic and inflammatory markers will be determined by specific antibody arrays. Our results indicate that fursultiamine prevents VEGF-induced endothelial cell growth in a dose-dependent manner. Further, fursultiamine prevents the in vitro tube formation as determined tube formation assays. Further, fursultiamine also regulated the VEGF-induced expression of various angiogenic markers such as VEGF, PDGF, EGF, Ang1, Ang2, angiogenin, and FGF, inflammatory markers such IL-1b, GM-CSF, TGF-b, MMPs, and others such as TIMPs, PTX3 and CF-III. Based on our in vitro data, we suggest that fursultiamine could prevent VEGF-induced angiogenesis, and this compound as a potent anti-angiogenic agent that could prevent cancer growth and metastasis. Citation Format: Sabrina Tran, Zachary Frost, Kota Ramana. Synthetic disulfide derivative of vitamin B1, fursultiamine, prevents VEGF-induced angiogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5385.

Isoimperatorin Inhibits Angiogenesis by Suppressing VEGFR2 Signaling Pathway.

Angiogenesis involves in many pathological processes, including tumor metastasis, diabetic retinopathy, and rheumatoid arthritis. Therefore, identifying therapeutic drugs that target angiogenesis may be a promising strategy for disease treatment. Isoimperatorin is a furanocoumarin with anti-inflammatory and anti-microbial effects. However, the impacts of isoimperatorin on angiogenesis and its underlying mechanisms remain unclear. This study aimed to verify its effects on vascular endothelial growth factor (VEGF)-induced endothelial angiogenesis. We employed various assays including 5-ethynyl-2'-deoxyuridine incorporation assay, transwell migration assay, wound healing assay, tube formation assay, and Western blot to evaluate the effects of isoimperatorin on angiogenesis in vitro. Additionally, we utilized Western blot and immunofluorescence analysis to examine the activation of vascular endothelial growth factor receptor (VEGFR) 2 and its downstream signaling pathways following isoimperatorin treatment. To further validate the anti-angiogenic effects of isoimperatorin in vivo, we conducted a matrigel plug assay and established an orthotopic tumor model. We demonstrated that pretreatment with isoimperatorin inhibited VEGF-induced endothelial cell proliferation, migration, and tube formation. Isoimperatorin also suppressed angiogenesis in vivo in a matrigel plug assay and in an orthotopic tumor model. Our results revealed that isoimperatorin exhibited anti-angiogenic effects via inhibiting VEGFR2 and its downstream signaling pathways activation. Our study showed that isoimperatorin suppressed angiogenesis by targeting the VEGFR2 signaling pathway and could be a potential therapeutic agent for targeting angiogenesis.

Inhibition of PDGFRβ alleviates endothelial cell apoptotic injury caused by DRP-1 overexpression and mitochondria fusion failure after mitophagy

Kawasaki disease (KD), described as “mucocutaneous lymph node syndrome”, affects infants and toddlers. Patients with KD suffer from an inflammatory cascade leading to vasculitis with a predilection for coronary arteries. While the symptoms and pathogenesis of KD have received more and more attention, the precise mechanisms are still debated. Researches show that endothelial dysfunction process in KD leads to arterial damage and affect clinical outcome. In this study, we constructed a Candida albicans water soluble fraction (CAWS)-induced KD murine model and penetrated investigating the mechanisms behind endothelial dysfunction. CAWS-induced mice presented remarkably elevated vascular endothelial cell growth factor (VEGF) levels. Abundant expression of VEGF was documented in all vessels that showed edema from acute KD. It has been reported that Platelet-derived growth factor (PDGF) co-expression normalizes VEGF-induced aberrant angiogenesis. Hyperexpression of PDGFRβ was induced in the thickened medial layer and vascular endothelium of KD mice. Masitinib (Mas) is an oral tyrosine kinase inhibitor of numerous targets, which can selectively target PDGFR signaling. We set out to explore whether Mas could regulate coronary pathology in KD. Mas administration significantly reduced the VEGF-induced endothelial cells migration. NOX4 was activated in vascular endothelial cells to produce more ROS. Mitochondrial dysregulated fission and mitophagy caused by DRP-1 overexpression precipitated the arterial endothelial cells injury. Here, mitophagy seemed to work as the driving force of DRP-1/Bak/BNIP3-dependent endothelial cells apoptosis. In summary, how mitophagy is regulated by DRP-1 under pathologic status is critical and complex, which may contribute to the development of specific therapeutic interventions in cardiovascular diseases patients, for example Masatinib, the inhibitor of PDGFRβ.Facts and questionsKawasaki disease causing systemic vasculitis, affects infants and toddlers.Coronary artery injury remains the major causes of morbidity and mortality.DRP-1 overexpression induces DRP-1/Bak/BNIP3-dependent endothelial cells apoptosis.PDGFRβ was high-expressed in the thickened medial layer of CAWS-induced KD mice.Inhibition of PDGFRβ signaling alleviates arterial endothelial cells injury.

HDAC6 and ERK/ADAM17 Regulate VEGF-Induced NOTCH Signaling in Lung Endothelial Cells.

Angiogenesis plays a critical role in various physiological and pathological processes and is regulated by VEGF. Histone Deacetylase 6 (HDAC6) is a class IIB HDAC that regulates cytoplasmic signaling through deacetylation and is emerging as a target for modulating angiogenesis. We investigated the hypothesis that VEGF-induced endothelial cell (EC) NOTCH signaling is regulated by HDAC6 through acetylation of NOTCH intracellular cytoplasmic domain (NICD). In pulmonary endothelial cells (EC), VEGF-induced activation of the NICD transcriptional response was regulated by ERK1/2 and ADAM 17 and required DLL4. While HDAC6 inhibition induced the acetylation of NICD and stabilized NICD, it repressed NICD-SNW1 binding required for the NOTCH transcriptional responses. In vitro experiments showed that HDAC6 inhibition inhibited lung EC angiogenesis, and neonatal mice treated with a systemic HDAC6 inhibitor had significantly altered angiogenesis and alveolarization. These findings shed light on the role of HDAC6 in modulating VEGF-induced angiogenesis through acetylation and repression of the transcriptional regulators, NICD and SNW1.

In Silico and In Vitro Evaluation of Bevacizumab Biosimilar MB02 as an Antitumor Agent in Canine Mammary Carcinoma.

Canine mammary carcinomas (CMC) are associated with major aggressive clinical behavior and high mortality. The current standard of care is based on surgical resection, without an established effective treatment scheme, highlighting the urgent need to develop novel effective therapies. Vascular endothelial growth factor (VEGF) is a key regulator of tumor angiogenesis and progression in the majority of solid cancers, including human and canine mammary carcinomas. The first therapy developed to target VEGF was bevacizumab, a recombinant humanized monoclonal antibody, which has already been approved as an anticancer agent in several human cancers. The goal of this work was to establish the therapeutic value of MB02 bevacizumab biosimilar in CMC. First, through different in silico approaches using the MUSCLE multiple-sequence alignment tool and the FoldX protein design algorithm, we were able to predict that canine VEGF is recognized by bevacizumab, after showing an extremely high sequence similarity between canine and human VEGF. Further, by using an ELISA-based in vitro binding assay, we confirmed that MB02 biosimilar was able to recognize canine VEGF. Additionally, canine VEGF-induced microvascular endothelial cell proliferation was inhibited in a concentration-dependent manner by MB02 biosimilar. These encouraging results show a high potential for MB02 as a promising therapeutic agent for the management of CMC.

Lentiviral delivered aflibercept OXB-203 for treatment of neovascular AMD

Neovascular age-related macular degeneration (nAMD) is a leading cause of blindness in the aging population, with vascular endothelial growth factor (VEGF) playing a key role. Treatment with recombinant anti-VEGFs is the current standard of care; however, it is only effective for 1-2months at a time and requires re-administration. Gene therapy could pave the way for stable, long-term expression of therapeutic anti-VEGF with a single dose, reducing the frequency of treatment and potentially improving clinical outcomes. As such, we have developed OXB-203, a lentiviral-based gene therapy encoding the anti-VEGF protein aflibercept. Aflibercept derived from OXB-203 exhibited comparable invitro binding characteristics to VEGF as recombinant aflibercept. Furthermore, its biological potency was demonstrated by the equivalent inhibition of VEGF-induced human umbilical vein endothelial cell (HUVEC) proliferation and tubule formation as recombinant aflibercept. In a rat choroidal neovascularization (CNV) model of nAMD, a single subretinal administration of OXB-203 reduced laser-induced CNV lesion areas analogous to an intravitreal bolus of recombinant aflibercept. Finally, in a head-to-head comparative study, aflibercept derived from OXB-203 was shown to be expressed at significantly higher levels in ocular tissues than from an AAV8-aflibercept vector following a single subretinal delivery to rats. These findings support the therapeutic potential of OXB-203 for the management of nAMD.

Efficiency co-delivery of ellagic acid and oxygen by a non-invasive liposome for ameliorating diabetic retinopathy

Diabetic retinopathy (DR) is one of the serious complications of diabetes, which has become the fourth leading cause of vision loss worldwide. Current treatment of DR relies on intravitreal injections of antiangiogenic agents, which has made considerable achievements in reducing visual impairment. However, long-term invasive injections require advanced technology and can lead to poor patient compliance as well as the incidence of ocular complications including bleeding, endophthalmitis, retinal detachment and others. Hence, we developed non-invasive liposomes (EA-Hb/TAT&isoDGR-Lipo) for efficiency co-delivery of ellagic acid and oxygen, which can be administered intravenously or by eye drops. Among that, ellagic acid (EA), as an aldose reductase inhibitor, could remove excessive reactive oxygen species (ROS) induced by high glucose for preventing retinal cell apoptosis, as well as reduce retinal angiogenesis through the blockage of VEGFR2 signaling pathway; carried oxygen could ameliorate DR hypoxia, and further enhanced the anti-neovascularization efficacy. Our results showed that EA-Hb/TAT&isoDGR-Lipo not only effectively protected retinal cells from high glucose-induced damage, but also inhibited VEGF-induced vascular endothelial cells migration, invasion, and tube formation in vitro. In addition, in a hypoxic cell model, EA-Hb/TAT&isoDGR-Lipo could reverse retinal cell hypoxia, thereby reducing the expression of VEGF. Significantly, after being administered as an injection or eye drops, EA-Hb/TAT&isoDGR-Lipo obviously ameliorated the structure (central retinal thickness and retinal vascular network) of retina by eliminating ROS and down-regulating the expression of GFAP, HIF-1α, VEGF and p-VEGFR2 in a DR mouse model. In summary, EA-Hb/TAT&isoDGR-Lipo holds great potentials in improvement of DR, which provides a novel approach for the treatment of DR.

Abstract 5080: TU2218, a novel ALK5/VEGFR2 dual inhibitor, overcomes tumor endothelial cell anergy and enhances anti-PD1 immunotherapy efficacy

Abstract Immune tolerance by TGF-β and VEGF is inextricably related with poor outcomes of approved anti-PD-(L)1 therapy. Accordingly, a dual target for ALK5 and VEGFR2 via single or combination treatments can be an unequivocal tactic to tune tumor-microenvironment (TME) favorable to ICI, and to essentially overcome immune evasion against TGF-β- and VEGF-enriched tumors. Specifically, several reports from clinical data suggest that VEGF-induced endothelial cell anergy (ECA) acts as a vascular immune checkpoint in TME immune response, and the activation of ECA is associated with worse outcomes. Herein, we demonstrate that TU2218, a first-in-class, orally available inhibitor against ALK5 and VEGFR2 can recover the downregulated endothelial adhesion molecules, i.e., ICAM-1 and VCAM-1, and suppress ECA. In this work, TU2218 completely recovered the expression of ICAM-1 and VCAM-1 on VEGF-induced ECA in HUVECs. The restored level of ICAM-1 and VCAM-1 at 1 μM TU2218 was equivalent to the activity of combined treatment of 1 μM Vactosertib (ALK5 inhibitor) and 25 μg/ml Ramucirumab (VEGFR2 inhibitor). 1 μM of Vactosertib alone, however, did not show such restoration. These results indicate that VEGF-induced ECA is mediated by both VEGFR2 and TGF-β signal, thereby validating the superiority of dual target strategy for ALK5 and VEGFR2 over a single target in overcoming ECA. We further tested if TU2218 could restore VEGF-induced decrease of Jurkat adhesion to HUVECs, considering the close relationship between the expression of adhesion-molecules of endothelial cell surface and the adhesion of lymphocytes to endothelium. TU2218 recovered the number of Jurkat adhering to VEGF-elicited HUVEC monolayer in a dose-dependent manner, but Vactosertib did not. Furthermore, the activity of TU2218 on Jurkat adhesion was reversed by VCAM-1 neutralizing antibody. Therefore, our results demonstrate that TU2218 improves Jurkat adhesion by restoring VCAM-1 expression. Finally, the in vivo translatability of TU2218 in overcoming ECA was confirmed with B16F10-bearing mice, a well-defined immune desert model, after treatments of anti-PD1 antibody, TU2218, or combined regimen for 15 days. TU2218 combined with an anti-PD1 antibody significantly suppressed tumor growth by c.a. 74 % compared to vehicle, thus being superior to a single treatment (e.g., tumor growth inhibition (TGI) 44% for TU2218, TGI 45% for anti-PD1). In this combination, TU2218 increased the number of both CD31+VCAM-1+ and IFNγ+CD8+ T cells in the tumor. We conclude that TU2218 leads not only to the enhancement of T cell-traffic toward TME, but also to the conversion of immune balance favorable to anti-PD1 therapy. The Phase 1b trial of TU2218 combined with pembrolizumab is underway for advanced solid cancers (NCT05204862). Citation Format: Jihyun Lee, Nam-hoon Kim, Hun-Taek Kim. TU2218, a novel ALK5/VEGFR2 dual inhibitor, overcomes tumor endothelial cell anergy and enhances anti-PD1 immunotherapy efficacy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5080.

Fruquintinib/HMPL-013 ameliorates cognitive impairments and pathology in a mouse model of cerebral amyloid angiopathy (CAA)

Cerebral amyloid angiopathy (CAA) is characterized by the cerebrovascular amyloid-β (Aβ) accumulation, and always accompanied by Alzheimer's disease (AD). The mechanisms revealing CAA pathogenesis are still unclear, and it is challenging to develop an efficient therapeutic strategy for its treatment. Vascular endothelial growth factor (VEGF) and its receptors including VEGFR-1,-2,-3 activation are involved in Aβ processing, and modulate numerous cellular events associated with central nervous system (CNS) diseases. In the present study, we attempted to explore the regulatory function of fruquintinib (also named as HMPL-013), a highly selective inhibitor of VEGFR-1,-2,-3 tyrosine kinases, on CAA progression in Tg-SwDI mice. Here, we found that HMPL-013-rich diet consumption for 12 months significantly improved the behavioral performances and cerebral blood flow (CBF) of Tg-SwDI mice compared with the vehicle group. Importantly, HMPL-013 administration considerably reduced Aβ1-40 and Aβ1-42 burden in cortex and hippocampus of Tg-SwDI mice through regulating Aβ metabolism process. Congo red staining confirmed Aβ deposition in vessel walls, reflecting CAA formation, which was, however, strongly ameliorated after HMPL-013 treatment. Neuron death, aberrant glial activation and pro-inflammatory response in brain tissues of Tg-SwDI mice were dramatically alleviated after HMPL-013 consumption. More studies showed that the protective effects of HMPL-013 against CAA might be partially attributed to its regulation on the expression of genes associated with blood vasculature. Intriguingly, VEGF and phosphorylated VEGFR-1,-2 protein expression levels were remarkably decreased by HMPL-013 in cortex and hippocampus of Tg-SwDI mice, which were validated in HMPL-013-treated brain vascular endothelial cells (BVECs) under hypoxia. Finally, we found that VEGF-induced human umbilical vein endothelial cells (HUVEC) proliferation and tube formation were strongly abolished upon HMPL-013 exposure. Collectively, all these findings demonstrated that oral administration of HMPL-013 had therapeutic potential against CAA by reducing Aβ deposition, inflammation and neuron death via suppressing VEGF/VEGFR-1,-2 signaling.

Abstract 12127: Protein Disulfide Isomerase A1 as Novel Redox Sensor in VEGFR2 Signaling and Angiogenesis

Background: VEGFR2 signaling in endothelial cells (ECs) is regulated by reactive oxygen species (ROS) derived from NADPH oxidases (NOXs) and mitochondria, which plays an important role in postnatal angiogenesis. However, it remains unclear how highly diffusible ROS signal enhances VEGFR2 signaling and reparative angiogenesis. Protein disulfide isomerase A1 (PDIA1) functions as an oxidoreductase depending on the redox environment. However, role of PDIA1 in ROS-dependent VEGFR2 signaling and angiogenesis is entirely unknown. Results: Here we showed that PDIA1 co-immunoprecipitated with VEGFR2 or colocalized with either VEGFR2 or early endosome marker Rab5, but not late endosome marker Rab7, at the perinuclear region in human ECs in response to VEGF. PDIA1 silencing significantly reduced VEGF-induced EC migration (43.3%), proliferation (52.8%) and spheroid sprouting via inhibiting VEGFR2 signaling. Mechanistically, VEGF stimulation rapidly increased Cys-OH formation of PDIA1 via the NOX4-mitochondrial ROS axis. Experiments using “redox-dead” mutant PDIA1 with replacement of the active four Cys residues with Ser revealed that VEGF-induced PDIA1 CysOH formation promoted angiogenic responses by increasing phosphorylation of VEGFR2 at Y1175 via oxidative inactivation of PTP1B. In vivo, Pdia1+/- mice showed impaired angiogenesis in developmental retina and Matrigel plug models as well as ex vivo aortic ring sprouting model. Hindlimb ischemia model revealed that PDIA1 expression was markedly increased in angiogenic CD31 positive ECs of ischemic muscles, and that limb perfusion recovery and neovascularization (48%) in response to ischemic injury were significantly impaired in EC-specific Pdia1 conditional knockout mice. Conclusion: PDIA1 can sense VEGF-induced H2O2 signal via CysOH formation to promote VEGFR2 signaling and angiogenesis in ECs via oxidative inactivation of PTP1B, thereby enhancing postnatal angiogenesis. The oxidized PDIA1 is a potential therapeutic target for treatment of ischemic vascular diseases

Protein disulfide isomerase A1 as a novel redox sensor in VEGFR2 signaling and angiogenesis.

VEGFR2 signaling in endothelial cells (ECs) is regulated by reactive oxygen species (ROS) derived from NADPH oxidases (NOXs) and mitochondria, which plays an important role in postnatal angiogenesis. However, it remains unclear how highly diffusible ROS signal enhances VEGFR2 signaling and reparative angiogenesis. Protein disulfide isomerase A1 (PDIA1) functions as an oxidoreductase depending on the redox environment. We hypothesized that PDIA1 functions as a redox sensor to enhance angiogenesis. Here we showed that PDIA1 co-immunoprecipitated with VEGFR2 or colocalized with either VEGFR2 or an early endosome marker Rab5 at the perinuclear region upon stimulation of human ECs with VEGF. PDIA1 silencing significantly reduced VEGF-induced EC migration, proliferation and spheroid sprouting via inhibiting VEGFR2 signaling. Mechanistically, VEGF stimulation rapidly increased Cys-OH formation of PDIA1 via the NOX4-mitochondrial ROS axis. Overexpression of "redox-dead" mutant PDIA1 with replacement of the active four Cys residues with Ser significantly inhibited VEGF-induced PDIA1-CysOH formation and angiogenic responses via reducing VEGFR2 phosphorylation. Pdia1+/- mice showed impaired angiogenesis in developmental retina and Matrigel plug models as well as ex vivo aortic ring sprouting model. Study using hindlimb ischemia model revealed that PDIA1 expression was markedly increased in angiogenic ECs of ischemic muscles, and that ischemia-induced limb perfusion recovery and neovascularization were impaired in EC-specific Pdia1 conditional knockout mice. These results suggest that PDIA1 can sense VEGF-induced H2O2 signal via CysOH formation to promote VEGFR2 signaling and angiogenesis in ECs, thereby enhancing postnatal angiogenesis. The oxidized PDIA1 is a potential therapeutic target for treatment of ischemic vascular diseases.

LncRNA Meg3 knockdown reduces corneal neovascularization and VEGF-induced vascular endothelial angiogenesis via SDF-1/CXCR4 and Smad2/3 pathway

The crucial effect of vascular endothelial growth factor (VEGF)-induced vascular angiogenesis has been well known in corneal neovascularization (CNV). This research aimed to determine the underlying value and mechanism of Meg3 on CNV in vivo and in vitro. In an alkali-burned mouse model, length and area of new vessels were increased along with thinning of corneal epithelium, accompanied by the overexpression of Meg3. Notably, subconjunctival injection of shMeg3 suppressed the degree of injury in cornea, causing expression of the angiogenesis markers--VEGF-A and CD31 decreased. In VEGF-induced human umbilical vein endothelial cells (HUVECs), knockdown of Meg3 antagonized the enhancement of viability, proliferation, wound healing ability and angiogenesis by VEGF. The proteins expression of VEGF-A, CD31, SDF-1/CXCR4 as well as phosphoraylation-Smad2/3 pathways, which were related to angiogenesis, were reduced with Meg3 deficiency. Overall, knockdown of Meg3 alleviated formation of neovascularization in alkali-burned corneas and reduced VEGF-induced angiogenesis by inhibiting SDF-1/CXCR4 and Smad2/3 signaling in vitro.

Chlorinated benzothiadiazines inhibit angiogenesis through suppression of VEGFR2 phosphorylation

Angiogenesis inhibitors are a critical pharmacological tool for the treatment of solid tumors. Suppressing vascular permeability leads to inhibition of tumor growth, invasion, and metastatic potential by blocking the supply of oxygen and nutrients. Disruption of the vascular endothelial growth factor (VEGF) signaling pathway is a validated target for the design of antiangiogenic agents. Several VEGFR2 inhibitors have been clinically approved over the past years. Structural analysis of these clinical VEGFR2 inhibitors highlighted key functional group overlap with the benzothiadiazine core contained in a library of in-house compounds. Herein we ascribe anti-angiogenic activity to a series of chlorinated benzothiadiazines. Selected compounds show significant activity to completely ameliorate VEGF-induced endothelial cell proliferation by suppression of VEGFR2 phosphorylation. The scaffold is devoid of activity to inhibit carbonic anhydrases and generally lacks cytotoxicity across a range of cancer and non-malignant cell lines. Assay of activity at 468 kinases shows remarkable selectivity with only four kinases inhibited > 65% at 10 µM concentration, and with significant activity to inhibit TNK2/ACK1 and PKRD2 by > 90%. All four identified kinase targets are known modulators of angiogenesis, thus highlighting compound 17b as a novel angiogenesis inhibitor for further development.

Malaysian Tualang Honey Suppresses the Angiogenic Events in Endothelial Cells Induced by Vascular Endothelial Growth Factor

Anti-VEGF therapy has been used as the anti-angiogenic agent in cancer treatment. However, the treatment often couples with severe complications. Complementary natural products that offer similar anti-angiogenic potency with less side effects are sought as alternatives. Study showed that Malaysian Tualang Honey (MTH) can inhibit inflammation-induced vascular hyperpermeability in vitro and in vivo, but the effect on the angiogenesis process remains unclear. Thus, this study aims to determine the anti-angiogenic effects of MTH. Effects of MTH ranging from 0.3% to 0.9% on VEGF-induced human umbilical vein endothelial cells (HUVEC) angiogenesis was determined by proliferation, migration and tube-formation assays. Matrix metalloproteinase-2 (MMP-2) secretion from HUVEC and VEGF production from MCF7 cancer cells in response to MTH were also quantified by using ELISA kits. Suramin, an angio-suppressive agent was used as positive control. MTH significantly suppressed HUVEC proliferation (from 155% proliferation rate to 54%); migration (~ 50% inhibition rate) and tube-formation (69% reduction) induced by VEGF. These findings were explained by the significant reduction (p < 0.05) of VEGF-induced MMP-2 secretion in HUVEC with 39% suppression exhibited by 0.9% of MTH. MTH also significantly (p < 0.05) reduced VEGF secretion (19% reduction compared to control) in MCF7 breast cancer cells. Our findings suggest that the anti-angiogenic effects of MTH mainly targets endothelial cell through inhibition cell proliferation, migration and tube formation capacity, via suppression of MMP-2 secretion by the endothelial cells. However, MTH has less prominent effect on suppressing VEGF secretion by the MCF7 cancer cells.

Bioactive fractions and compound of Ardisia crispa roots exhibit anti-arthritic properties mediated via angiogenesis inhibition in vitro

BackgroundArdisia crispa (Thunb.) A.DC (Primulaceae), is a medicinal herb traditionally used by Asian people as remedies to cure inflammatory related diseases, including rheumatism. The plant roots possess various pharmacological activities including antipyretic, anti-inflammation and antitumor. Previous phytochemical studies of the plant roots have identified long chain alkyl-1,4-benzoquinones as major constituents, together with other phytochemicals. Hexane fraction of the plant roots (ACRH), was previously reported with anti-angiogenic and anti-arthritic properties, while its effect on their anti-arthritic in vitro, is yet unrevealed. Considering the significance of angiogenesis inhibition in developing new anti-arthritic agent, thus we investigated the anti-arthritic potential of Ardisia crispa roots by suppressing angiogenesis, in vitro.MethodsArdisia crispa roots hexane extract (ACRH) was prepared from the plant roots using absolute n-hexane. ACRH was fractionated into quinone-rich fraction (QRF) and further isolated to yield benzoquinonoid compound (BQ), respectively. In vitro experiments using VEGF-induced human umbilical vein endothelial cells (HUVECs) and IL-1β-induced human fibroblast-like synoviocytes for rheumatoid arthritis (HFLS-RA) were performed to evaluate the effects of these samples on VEGF-induced HUVECs proliferation and tube formation, and towards IL-1β-induced HFLS-RA proliferation, invasion, and apoptosis, respectively. Therapeutic concentrations (0.05, 0.5, and 5 μg/mL) tested in this study were predetermined based on the IC50 values obtained from the MTT assay.ResultsACRH, QRF, and BQ exerted concentration-independent antiproliferative effects on VEGF-induced HUVECs and IL-1β-induced HFLS-RA, with IC50 values at 1.09 ± 0.18, 3.85 ± 0.26, and 1.34 ± 0.16 μg/mL in HUVECs; and 3.60 ± 1.38, 4.47 ± 0.34, and 1.09 ± 0.09 μg/mL in HFLS-RA, respectively. Anti-angiogenic properties of these samples were verified via significant inhibition on VEGF-induced HUVECs tube formation, in a concentration-independent manner. The invasiveness of IL-1β-induced HFLS-RA was also significantly inhibited in a concentration-independent manner by all samples. ACRH and BQ, but not QRF, significantly enhanced the apoptosis of IL-1β-induced HFLS-RA elicited at their highest concentration (5 μg/mL) (P < 0.05).ConclusionsThese findings highlight the bioactive fractions and compound from Ardisia crispa roots as potential anti-arthritic agents by inhibiting both HUVECs and HFLS-RA’s cellular functions in vitro, possibly mediated via their anti-angiogenic effects.

Inhibiting Endothelial Cell Function in Normal and Tumor Angiogenesis Using BMP Type I Receptor Macrocyclic Kinase Inhibitors.

Simple SummaryAnti-angiogenesis agents have shown anti-cancer activity by preventing blood vessel ingrowth, thereby limiting tumour growth and metastasis. Although these molecules lead to prolonged overall survival of cancer patients, therapy resistance is easily acquired. Therefore, novel inhibitors against other signaling pathways mediating angiogenesis are needed to achieve more efficient and sustainable targeting of the angiogenesis process. Here, we synthesized and identified two compounds belonging to a new class of small molecules termed macrocyclics that selectively inhibit bone morphogenetic protein receptor kinase activity. One compound also inhibits vascular endothelial growth factor-induced signalling. Treatment studies using in vitro cultured cells and zebrafish embryos revealed that both compounds impaired endothelial cell function and decreased normal and tumour-induced angiogenesis. Both compounds might provide a steppingstone for the development of novel-angiogenesis therapeutic agents.Angiogenesis, i.e., the formation of new blood vessels from pre-existing endothelial cell (EC)-lined vessels, is critical for tissue development and also contributes to neovascularization-related diseases, such as cancer. Vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs) are among many secreted cytokines that regulate EC function. While several pharmacological anti-angiogenic agents have reached the clinic, further improvement is needed to increase clinical efficacy and to overcome acquired therapy resistance. More insights into the functional consequences of targeting specific pathways that modulate blood vessel formation may lead to new therapeutic approaches. Here, we synthesized and identified two macrocyclic small molecular compounds termed OD16 and OD29 that inhibit BMP type I receptor (BMPRI)-induced SMAD1/5 phosphorylation and downstream gene expression in ECs. Of note, OD16 and OD29 demonstrated higher specificity against BMPRI activin receptor-like kinase 1/2 (ALK1/2) than the commonly used small molecule BMPRI kinase inhibitor LDN-193189. OD29, but not OD16, also potently inhibited VEGF-induced extracellular regulated kinase MAP kinase phosphorylation in ECs. In vitro, OD16 and OD29 exerted strong inhibition of BMP9 and VEGF-induced ECs migration, invasion and cord formation. Using Tg (fli:EGFP) zebrafish embryos, we found that OD16 and OD29 potently antagonized dorsal longitudinal anastomotic vessel (DLAV), intra segmental vessel (ISV), and subintestinal vessel (SIV) formation during embryonic development. Moreover, the MDA-MB-231 breast cancer cell-induced tumor angiogenesis in zebrafish embryos was significantly decreased by OD16 and OD29. Both macrocyclic compounds might provide a steppingstone for the development of novel anti-angiogenesis therapeutic agents.

Guanabenz and Clonidine, α2-Adrenergic Receptor Agonists, Inhibit Choroidal Neovascularization.

Neovascular age-related macular degeneration (AMD) with choroidal neovascularization (CNV) is a leading cause of blindness in elderly people. Anti-vascular endothelial growth factor (anti-VEGF)-drugs are used to treat AMD patients; however, some patients are resistant to these therapies. The purpose of this study was to investigate the anti-angiogenic effects of α2-adrenergic agonists, including guanabenz and clonidine. We evaluated the anti-angiogenic effects of α2-adrenergic agonists in human retinal microvascular endothelial cells (HRMECs). A proliferation assay was conducted, and the migration ratio was evaluated. In a laser-induced CNV model, guanabenz and clonidine were delivered via intraperitoneal injection or implantation of an osmotic pump device. Fourteen days following CNV induction, CNV lesion size and fundus fluorescein angiography (FFA) were evaluated. Guanabenz and clonidine inhibited VEGF-induced retinal endothelial cell growth and migration. In the CNV model mice, CNV lesion sizes were reduced by intraperitoneal administration of guanabenz or clonidine. Data, including body weight, systolic blood pressure, and heart rate showed that guanabenz (0.5 and 2.0 mg/kg/day) had little effect on these parameters; conversely, a high dose of clonidine (1.0 mg/kg/day) did affect these parameters. Additionally, clonidine did not affect CNV size, but continuous administration of guanabenz attenuated both CNV size and leakage from neovessels. Our study suggests a key role for α2-adrenergic receptors during CNV formation. Therefore, we suggest that α2-adrenergic receptor agonists may represent novel therapeutic drugs for patients with neovascular AMD.

The Antiangiogenic Effect of Sanguinarine Chloride on Experimental Choroidal Neovacularization in Mice via Inhibiting Vascular Endothelial Growth Factor.

Background: The purpose of this study is to investigate the antiangiogenic effect of Sanguinarine chloride (SC) on models of age-related macular degeneration (AMD) both in vivo and in vitro. Methods: Choroidal neovascularization (CNV) was conducted by laser photocoagulation in C57BL6/J mice. SC (2.5 μM, 2 μl/eye) was intravitreally injected immediately after laser injury. The control group received an equal amount of PBS. 7 days after laser injury, CNV severity was evaluated using fundus fluorescein angiography, hematoxylin and eosin (H&E) staining, and choroid flat-mount staining. Vascular endothelial growth factor (VEGF) expression in the retina/choroid complex was measured by western blot analysis and ELISA kit. In vitro, human retinal microvascular endothelial cells (HRMECs) were used to investigate the effects of SC on cell tube formation, migration, and cytotoxicity. The expression of VEGF-induced expression of extracellular signal-regulated kinase (ERK)1/2, protein kinase B (AKT), mitogen-activated protein kinases (p38-MAPK) in vitro and laser induced VEGF expression in vivo were also analyzed. Results: SC (≤2.5 μM) was safe both in vitro and in vivo. Intravitreal injection of SC restrained the formation of laser induced CNV in mice and decreased VEGF expression in the laser site of the retina/choroid complex. In vitro, SC inhibited VEGF-induced tube formation and endothelial cell migration by decreasing the phosphorylation of AKT, ERK1/2, and p38-MAPK in HRMECs. Conclusions: SC could inhibit laser-induced CNV formation via down-regulating VEGF expression and restrain the VEGF-induced tube formation and endothelial migration. Therefore, SC could be a potential candidate for the treatment of wet AMD.