VEGF Antagonists Research Articles

Reduction of pathological retinal neovascularization, vessel obliteration, and artery tortuosity by PEDF protein in an oxygen-induced ischemic retinopathy rat model.

Retinopathy of prematurity (ROP) is a severe retinal disease in premature infants characterized by pathological neovascularization, obliteration of retinal vessels and increased vessel tortuosity. Currently, there are no completely satisfactory treatments for ROP. Pigment epithelium-derived factor (PEDF), a potent inhibitor of angiogenesis, appears late in gestation and its deficiency may be linked to development of ROP. This study investigates the preclinical efficacy of PEDF protein alone or in combination with VEGF antagonists for treating ROP. The safety of PEDF protein in the rat eye was assessed using functional invivo measurements and histology. The efficacy of intravitreal injections (IVI) of various treatments was evaluated in a rat oxygen-induced retinopathy (OIR) model using invivo imaging and flatmount analyses. No functional or histological side-effects were found in rat eyes after intravitreal PEDF protein injection. PEDF protein alone or combined with anti-VEGF drugs significantly reduced pathological neovascularization and vessel obliteration, comparable to the effects of anti-VEGF drugs alone. Regarding arterial tortuosity, treatment with a combination of PEDF, and VEGF antagonist was more effective than treatment with anti-VEGF alone. IVI of PEDF protein is safe. PEDF protein alone or combined with VEGF antagonists shows similar efficacy in reducing pathological neovascularization and vessel obliteration as anti-VEGF agents. Furthermore, only treatments involving PEDF protein, alone or with VEGF antagonists, significantly improved the quality of retinal vasculature. Thus, PEDF protein alone or combined with anti-VEGF agents presents a promising alternative to current anti-VEGF treatments for ROP.

Identification of Potential Inhibitors from Medicinal Plant-based Phytochemicals for the Influential C4 Target of Diabetic Retinopathy by Molecular Docking Studies.

Diabetic retinopathy is the major cause of vision failure in diabetic patients, and the current treatment involves the practice of glucocorticoids or VEGF antagonists that are "off-label". A few small organic molecules against DR were discovered many years ago. Nutraceuticals are naturally available functional foods that endorse different health benefits, including vitamins, antioxidants, minerals, fatty acids, and amino acids that can defer the development of some diseases. Numerous studies reported that nutraceuticals encourage multiple therapeutic benefits and provide protection against various diseases. In diabetes, nutraceuticals contribute to improving insulin sensitivity, metabolism regulation, and lower hyperglycemia. The major aim of this study is to discover the most active drug from natural or plant sources. In this work, 42 phytochemical constituents from 4 kinds of plants were docked with the C4 target of diabetic retinopathy by an in silico molecular docking study. According to the binding energy, all the phytoconstituents possessed good to high attraction towards the target, and 6 phytochemicals, such as terchebulin, punicalagin, chebulagic acid, casuarinin, punicalin, and pedunculagin, disclosed superior binding energy towards the target than standard ruboxistaurin via the interactions of conventional hydrogen bonding, pi-alkyl interactions, etc. Molecular dynamic simulation studies further established the stability of the phytoconstituents, and ADMET studies proved the safety profile of these phytoconstituents. Hence, the current study suggested that the phytochemicals from various herbs inhibit the C4 target of diabetic retinopathy and can be utilized as lead compounds to develop analogs or repurposed for the treatment of DR.

Investigation of the Efficacy of Bevacizumab Treatment in An Experimental Rat Model of Chronic Subdural Hematoma

IntroductionChronic subdural hematoma (cSDH), a condition that develops over time, is characterized by inflammation, angiogenesis, and membrane development. As the population's average age increases, the incidence of cSDH is expected to grow. While surgery is the primary treatment technique, medicinal therapy options are being explored for high-risk patients. Currently, the most effective therapy combination is dexamethasone (Dex) and atorvastatin (Ato); however, it is associated with an increased risk of mortality. This study explored the effects of bevacizumab (Bev), a VEGF antagonist, on cSDH. Materials and MethodsNinety-five rats were divided into four groups (n = 18): sham, control hematoma, Dex-Ato, and Bev. Two separate autologous blood injections into the subdural space were used as the model. Weight was monitored for all rats to assess changes in their overall health. The control group was given i.p. saline, the Dex-Ato treatment was given by gavage, and the Bev treatment was given i.p. On 7th, 14th and 21st days six rats from each group were sacrificed and analyzed, while 23 rats were excluded from the experiment. ResultsThe maximum immunological response to cSDH was observed on day 14. Hematoma volume decreased over time in all groups. Dex-Ato and Bev were both found effective, while Dex-Ato caused weight loss. ConclusionBev had similar effects to the Dex-Ato group and was well tolerated by rats. Given that cSDH is a disease of the elderly and vulnerable populations, Bev may be a viable alternative that can shed light on the disease's etiology for future research.

A New Generation of Gene Therapies as the Future of Wet AMD Treatment.

Age-related macular degeneration (AMD) is an eye disease and the most common cause of vision loss in the Western World. In its advanced stage, AMD occurs in two clinically distinguished forms, dry and wet, but only wet AMD is treatable. However, the treatment based on repeated injections with vascular endothelial growth factor A (VEGFA) antagonists may at best stop the disease progression and prevent or delay vision loss but without an improvement of visual dysfunction. Moreover, it is a serious mental and financial burden for patients and may be linked with some complications. The recent first success of intravitreal gene therapy with ADVM-022, which transformed retinal cells to continuous production of aflibercept, a VEGF antagonist, after a single injection, has opened a revolutionary perspective in wet AMD treatment. Promising results obtained so far in other ongoing clinical trials support this perspective. In this narrative/hypothesis review, we present basic information on wet AMD pathogenesis and treatment, the concept of gene therapy in retinal diseases, update evidence on completed and ongoing clinical trials with gene therapy for wet AMD, and perspectives on the progress to the clinic of "one and done" therapy for wet AMD to replace a lifetime of injections. Gene editing targeting the VEGFA gene is also presented as another gene therapy strategy to improve wet AMD management.

Abstract B123: Mechanism of action of ivonescimab (AK112/SMT112): a first-in-class tetravalent Fc-silent bispecific antibody with dual blockade of PD-1 and VEGF that promotes cooperative biological effects

Abstract Background: Combination treatments using anti-PD-1/PD-L1 antibodies with other VEGF antagonists have shown enhanced clinical antitumor activities1. The expression of PD-1 and VEGF are found to be frequently upregulated and co-expressed in solid tumors. Importantly, VEGF promotes tumor angiogenesis and suppresses antitumor immune response2,3. Consequently, we characterized the mechanism-of-action of a novel first-in-class anti-PD-1/VEGF bispecific antibody, ivonescimab, designed to simultaneously inhibit PD-1-mediated immunosuppression and block tumor angiogenesis in the tumor microenvironment. Methods: Binding activity of ivonescimab to PD-1/VEGF were assessed by ELISA. Blockade of PD-1/VEGF signaling pathways was determined in luciferase reporter cell assays. Ivonescimab-VEGF complex formation was detected by SEC-HPLC. Cooperative binding of ivonescimab-VEGF complex to PD-1 or ivonescimab-PD-1 complex to VEGF was measured by Octet BLI. The enhanced PD-1 blockade bioactivity of ivonescimab with VEGF was evaluated in hPBMC and engineered cell-line co-culture/luciferase-reporter cell assays. Antitumor activity of ivonescimab was investigated in hPBMC-humanized SCID/Beige mice implanted with HCC827 (mEGFR lung adenocarcinoma) or U87MG (glioblastoma). Immuno-safety was assessed by FcγR binding, ADCC, ADCP assays, and reported clinical irAEs. Results: Ivonescimab displayed strong binding activity to human PD-1 and VEGF alone or simultaneously, effectively blocking interactions with ligands and the downstream signaling effects. In presence of VEGF, ivonescimab forms soluble complexes with VEGF dimers, leading to over 10-fold enhanced binding affinity (KD) of ivonescimab to PD-1. The avidity increase was consistent with reduced cell surface PD-1 expression on human T-cell lines, increased potency on blockade of PD-1/PD-L1 signaling and subsequent enhanced T cell activation in-vitro. Likewise, PD-1 binding enhanced ivonescimab binding to VEGF which was associated with enhanced VEGF-signaling blockade. Also, ivonescimab treatment demonstrated statistically significant dose-dependent antitumor response in hPBMC-humanized murine HCC827 and U87MG tumor models. Additionally, ivonescimab enhanced antitumor response in combination with anti-CD47 (AK117) or anti-CD73 (AK119) in mouse models. Finally, ivonescimab contains Fc-silencing mutations abrogating FcγRI/IIIa binding and showed significantly reduced ADCC, ADCP activities and cytokine release in-vitro. Clinically, this is consistent with the safety profile in Phase 1/2 studies of ivonescimab in advanced solid tumors4,5. Conclusions: Ivonescimab is a novel tetravalent anti-PD-1/VEGF bispecific antibody displaying unique cooperative binding to each of its intended targets consistent with increased in-vitro functional bioactivities compared with bevacizumab or PD-1 inhibitors alone. Importantly, the Fc-null IgG1 design resulted in reduced FcgR interactions and minimal ADCC, ADCP activities consistent with its clinical immunosafety profile. Ivonescimab is currently in Ph3 NSCLC trials in the US and EU. Citation Format: Tingting Zhong, Zhaoliang Huang, Xinghua Pang, Chunshan Jin, Xinrong He, Jose G Montoya, Betty Y Chang, Michelle Xia, Baiyong Li, Jing Min. Mechanism of action of ivonescimab (AK112/SMT112): a first-in-class tetravalent Fc-silent bispecific antibody with dual blockade of PD-1 and VEGF that promotes cooperative biological effects [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B123.

Mice with humanized immune system as novel models to study HIV-associated pulmonary hypertension.

People living with HIV and who receive antiretroviral therapy have a significantly improved lifespan, compared to the early days without therapy. Unfortunately, persisting viral replication in the lungs sustains chronic inflammation, which may cause pulmonary vascular dysfunction and ultimate life-threatening Pulmonary Hypertension (PH). The mechanisms involved in the progression of HIV and PH remain unclear. The study of HIV-PH is limited due to the lack of tractable animal models that recapitulate infection and pathobiological aspects of PH. On one hand, mice with humanized immune systems (hu-mice) are highly relevant to HIV research but their suitability for HIV-PH research deserves investigation. On another hand, the Hypoxia-Sugen is a well-established model for experimental PH that combines hypoxia with the VEGF antagonist SU5416. To test the suitability of hu-mice, we combined HIV with either SU5416 or hypoxia. Using right heart catheterization, we found that combining HIV+SU5416 exacerbated PH. HIV infection increases human pro-inflammatory cytokines in the lungs, compared to uninfected mice. Histopathological examinations showed pulmonary vascular inflammation with arterial muscularization in HIV-PH. We also found an increase in endothelial-monocyte activating polypeptide II (EMAP II) when combining HIV+SU5416. Therefore, combinations of HIV with SU5416 or hypoxia recapitulate PH in hu-mice, creating well-suited models for infectious mechanistic pulmonary vascular research in small animals.

Blocking of VEGF-A is not sufficient to completely revert its long-term effects on the barrier formed by retinal endothelial cells

The VEGF-A-induced functional impairment of the barrier formed by retinal endothelial cells (REC) can be prevented and even – at least temporarily – reverted by trapping the growth factor in a complex with a VEGF-binding protein or by inhibiting the activity of the VEGF receptor 2 (VEGFR2). In an approach to emulate the clinically relevant situation of constant exposure to effectors, we investigated (1) whether prolonged exposure to VEGF-A165 for up to six days results in a different type of disturbance of the barrier formed by immortalized bovine REC (iBREC) and (2) whether alterations of the barrier induced by VEGF-A165 can indeed be sustainably reverted by subsequent treatment with the VEGF-A-binding proteins ranibizumab or brolucizumab. As a measure of barrier integrity, the cell index (CI) of iBREC cultivated on gold electrodes was monitored continuously. CI values declined shortly after addition of the growth factor and then remained low for more than six days over which considerable amounts of both extra- and intracellular VEGF-A were measured. Interestingly, the specific VEGFR2 inhibitor nintedanib normalized the lowered CI when added to iBREC pre-treated with VEGF-A165 for one day, but failed to do so when cells had been exposed to the growth factor for six days. Expression of the tight junction (TJ) protein claudin-5 was unchanged early after addition of VEGF-A165 but higher after prolonged treatment, whereas decreased amounts of the TJ-protein claudin-1 remained low, and increased expression of the plasmalemma vesicle-associated protein (PLVAP) remained high during further exposure. After two days, the characteristic even plasma membrane stainings of claudin-1 or claudin-5 appeared weaker or disordered, respectively. After six days the subcellular localization of claudin-5 was similar to that of control cells again, but claudin-1 remained relocated from the plasma membrane. To counteract these effects of VEGF-A165, brolucizumab or ranibizumab was added after one day, resulting in recovery of the then lowered CI to normal values within a few hours. However, despite the VEGF antagonist being present, the CI declined again two days later to values that were just slightly higher than without VEGF inhibition during further assessment for several days. At this stage, neither the supernatants nor whole cell extracts from iBREC treated with VEGF-A165 and its antagonists contained significant amounts of free VEGF-A. Treatment of VEGF-A165-challenged iBREC with ranibizumab or brolucizumab normalized expression of claudin-1 and claudin-5, but not completely that of PLVAP. Interestingly, the characteristic VEGF-A165-induced relocalization of claudin-1 from the plasma membrane was reverted within one day by any of the VEGF antagonists, but reappeared despite their presence after further exposure for several days. Taken together, barrier dysfunction induced by VEGF-A165 results from deregulated para- and transcellular flow but the precise nature or magnitude of underlying changes on a molecular level clearly depend on the time of exposure, evolving into a stage of VEGF-A165-independent barrier impairment. These findings also provide a plausible explanation for resistance to treatment with VEGF-A antagonists frequently observed in clinical practice.

Immunotherapies for hepatocellular carcinoma and intrahepatic cholangiocarcinoma: Current and developing strategies.

Liver cancer including hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA) is the third leading cause of cancer-related deaths worldwide. HCC arises from hepatocyte or hepatic stem cells, while iCCA originates from biliary epithelial cells, and the respective biological context are very different. Despite screening programs, the diagnosis of liver cancer is in most cases made when curative treatments such as surgery or ablation are not possible. In 2020, after a decade of using only tyrosine kinase inhibitors (TKI), a combination of an immune-check point inhibitor (ICI) and a VEGF antagonist proved superior to a TKI as first line therapy of advanced HCC. In 2022, the addition of an ICI to standard chemotherapy demonstrated an improvement of patient survival in iCCA. Moreover, ICI offer an unprecedented rate of durable responses to HCC and iCCA patients. Nevertheless, still two thirds of patients do not respond to ICI-based combinations, and research efforts are focused on deciphering the mechanisms of immune evasion of these lethal cancers. Reliable predictive and prognostic biomarkers are still lacking, but the molecular phenotyping of the tumor microenvironment is currently providing potential candidates for patient stratification. In this review, we will summarize the current knowledge on the immune biology of the liver, the discovery of cell-intrinsic and immune cell-mediated mechanisms of immune evasion by means of high-resolution single cell data, the main targets of current immunotherapy approaches, and the recent milestones in immunotherapy of HCC and iCCA.

Abstract 14009: Longitudinal Analysis Supports Reproducibility Of Treatment With Sildenafil In The Semaxanib/Hypoxia Rat Model Of Pulmonary Arterial Hypertension

Rats develop translatable pulmonary arterial hypertension (PAH) after exposure to the VEGF antagonist semaxanib (SU5416) concurrent with hypoxia. Sildenafil is used clinically for PAH and as a positive control in this preclinical model. A longitudinal analysis was constructed by pooling data from discrete rat PAH studies conducted over an 8-year period to evaluate the stability and reproducibility of the SU5416/Hypoxia rodent model as well as the response to sildenafil. Sprague-Dawley rats were administered SU5416 on Day 1 and subsequently maintained in a low oxygen environment for 28 days. Sildenafil was given as a positive control in either prevention or treatment mode. Pulmonary hemodynamics were obtained on the final day as appropriate by design; hearts were also assessed for right ventricular hypertrophy. Prevention studies (≥17) were terminated following 28 days of hypoxia while most treatment studies (≥17) had a protocol-defined period of normoxia following hypoxia. In prevention studies, the mean of both systolic pulmonary arterial pressure (sPAP) and the right ventricular hypertrophy Fulton’s Index (FI) remained consistent across studies over the 7-year period: sPAP: PAH vehicle control (PAH-VC) 70 ± 19 mmHg (n = 190), sildenafil 51 ± 15 mmHg (n = 165), p<.05; FI: PAH-VC 0.5573 ± 0.1048 (n = 210), sildenafil 0.4984 ± 0.1007 (n = 172), p<.05. Sildenafil elicited mean reductions in sPAP of 28% and FI of 11% over the 7-year period. Mean survival rate was comparable for PAH-VC (96%) and sildenafil groups (99%).In treatment studies, the mean of both sPAP and FI remained consistent across studies over the 8-year period: sPAP: PAH-VC 89 ± 24 mmHg (n = 148) vs. sildenafil 67 ± 22 mmHg (n = 168), p<.05 vs PAH-VC; FI: PAH-VC 0.5938 ± 0.0944 (n = 168) vs. sildenafil 0.5336 ± 0.1077 (n = 173), p<.05 vs PAH-VC. Sildenafil elicited mean reductions in sPAP of 25% and FI of 10% over the 8-year period. Mean survival rate was comparable for PAH-VC (91%) and sildenafil groups (95%).The effects of sildenafil on reduction of PAH were stable and of similar magnitude in both prevention and treatment studies over an 8-year period. Thus, this longitudinal analysis indicates a reproducible and consistent effect of both the SU5416/Hypoxia PAH model and the use of sildenafil as a positive control.

Ten-Year Outcomes of Intravitreal Bevacizumab for Myopic Choroidal Neovascularization: Analysis of Prognostic Factors.

The current standard treatment of myopic choroidal neovascularisation (mCNV) is intravitreal injection of VEGF antagonists. This study was proposed to assess efficacy and safety of intravitreal bevacizumab (IVB) for the treatment of mCNV across a 10-year follow-up. Thirty eyes of thirty patients with treatment-naïve mCNV who underwent IVB and were followed up with for a minimum of ten years were recruited for the present retrospective cohort study. All participants were treated with three monthly IVB at baseline and then evaluated and treated under pro re nata (PRN) schedule. Outcome measures were to determine BCVA changes over years and identify the predictive factors of both final visual outcome and need for retreatment. Analysis of the main involved prognostic factors with correlations among variables is reported. Visual acuity remained stable at 10-year follow-up (p = 0.001) with the greatest improvement at 2 years (p < 0.0001) in all CNV locations. Baseline BCVA correlated positively with final BCVA (β = 0.88, p < 0.0001, R2: 0.75). No predictive factors for the need of additional injections were identified. Retinal and choroidal thickness significantly reduced over time but without correlation with the number of injections. CNV max height and area significantly decreased at 10 years (p < 0.0001 and p = 0.003, respectively), with complete regression of mCNV lesion in 40% of subjects. Intravitreal bevacizumab resulted as long-term effective and safe therapy for mCNV with sustained results at 10 years.

Role of WNT Agonists, BMP and VEGF Antagonists in Rescuing Osteoarthritic Knee Cartilage in a Rat Model.

The superficial zone of articular cartilage (AC) is vital for its function and biomechanics. The damaged AC gets vascularized and undergoes hypertrophy and ossification. Studies have highlighted these two as the major causative factors in osteoarthritis (OA). We aimed at preventing the OA progression in a rat knee instability model by inhibiting the vascular ingrowth and ossification using VEGF and BMP antagonist. A WNT agonist was also used to promote AC regeneration because of its protective effect on the superficial layer. Rat knee OA was created by surgical excision of the medial meniscus and medial collateral ligament. Forty rats were divided into two groups of twenty each for surgical control and tests (surgery + intra-articular injection of drugs every two weeks). Ten animals from each group were sacrificed at four and eight weeks. Histology was mainly used to evaluate the outcome. A surgical OA model was successfully created with higher histological scores for operated knees, both in short- (P = 0.0001) and long-term (P = 0.001). Modified Mankin score was lesser in the test animals as compared to control (P = 0.17) in the short-term, but the trend was reversed in the long-term (P = 0.13). Subgroup analysis revealed that repeated injections in the anterolateral compartment contributed to higher scores in the lateral (P = 0.03) and anterior (P = 0.03) compartment of the knee in the long-term. The combinatorial approach was effective in controlling the OA in short-term. Further studies are needed to test the sustained drug delivery system to improve the outcome.

Structural Studies on an Anti-Angiogenic Peptide Using Molecular Modeling.

Background: Development of VEGF antagonists, which inhibit its interaction with the receptors, is a widely used strategy for the inhibition of angiogenesis and tumor growth.Objectives: In the present study, a VEGFR-1 antagonistic peptide was designed and its potential for binding to VEGFR-1 and VEGFR-2 was evaluated by theoretical studies.Materials and Methods: Based on the X-ray structure of VEGF-B/VEGFR-1 D2 (PDB ID: 2XAC), an antagonistic peptide (known as VGB1) was designed, and its model structure was constructed using homology modeling in the MODELLER, version 9.16. The validity of the modeled structures was estimated employing several web tools. Finally, one model was chosen and molecular dynamics (MD) simulation was applied using the GROMACS package, version 5.1.4, to allow conformational relaxation of the structure. Next, docking process of the peptide with VEGFR-1 and VEGFR-2 was performed by HADDOCK web server and the docking structures were optimized by MD simulation for 20 ns. The far-UV circular dichroism (CD) spectrum of VGB1 was recorded to evaluate the overall structure of the peptide. Results: The far-UV CD spectrum indicated that VGB1 contains α helix structure. The results from docking studies suggested that Van der Waals and nonpolar interactions play the most important role in the peptide binding to VEGFR-1. In addition, our results implicated the relevance of both Van der Waals and electrostatic interactions in the formation of complex between VGB1 and VEGFR-2. In addition to the common binding residues in the corresponding region of VEGF-A and VEGF-B, additional binding residues also were predicted for the interaction of VGB1 with VEGFR-1 and VEGFR-2.Conclusions: The results of MD and molecular docking simulations predicted that VGB1 recognizes both VEGFR-1 and VEGFR-2, which may lead to the prevention of the downstream signaling triggered by these receptors.

Targeted Antagonism of Vascular Endothelial Growth Factor Reduces Mortality of Mice with Acute Respiratory Distress Syndrome.

SummaryAcute respiratory distress syndrome (ARDS) is associated with a mortality of 45%. Our previous research indicated that anti-vascular endothelial growth factor (VEGF) could maintain the normal structure and function of the respiratory barrier. However, systemic application of VEGF antagonists would lead to animal death. This study attempts to study the targeted drug delivery for ARDS. In this study, we used soluble fms-like tyrosine kinase-1 (sFlt)-targeted ultrasound microbubbles to antagonize the effect of VEGF on lung tissue. Ninety male BALB/c mice were randomly assigned to 6 groups: phosphate buffer saline (PBS) group (PBS+PBS); blank group (PBS+empty microbubbles); lipopolysaccharide (LPS) group (LPS+PBS); ARDS group (LPS+empty microbubbles); control group (PBS+sFlt microbubbles); and treatment group (LPS+sFlt microbubbles). After administration of LPS or PBS in the corresponding groups, the sFlt-targeted microbubbles or empty microbubbles were injected into the blood circulation. Then the lungs were irradiated with ultrasound, which ruptured the drug-loaded microbubbles and helped release drugs to the lung tissues targeted. The lung injury score, lung wet/dry ratio (W/D), liver and kidney functions, and the mortality of the mice in all groups were investigated at the predetermined time point. The difference in mortality between groups was examined by Fisher test. Other data were analyzed by one-way analysis of variance (ANOVA). A value of P<0.05 indicates that the difference was significant. The results showed that the PaO2 levels were normal in the PBS group, the blank group, and the control group. The LPS group and ARDS group showed significant hypoxia. PaO2 was improved significantly in the treatment group. The lung injury score and W/D were normal in the PBS group, the blank group, and the control group. The lung injury score and W/D increased significantly in the LPS group and ARDS group and decreased in the treatment group (P<0.05). The mortality rate of the ARDS model was 60% (95% confidence interval 47.5%–72.5%), and that with sFlt-targeted microbubbles was significantly lower at only 40% (95% confidence interval 27.5%–52.5%, P<0.05). It was concluded that anti-VEGF with sFlt targeted ultrasound microbubbles attenuated the lung injury and ultimately reduced the 7-day mortality effectively. It might be a suitable therapeutic tool for the treatment of ARDS.

Endothelial HIF-2α as a Key Endogenous Mediator Preventing Emphysema.

Rationale: Endothelial injury may provoke emphysema, but molecular pathways of disease development require further discernment. Emphysematous lungs exhibit decreased expression of HIF-2α (hypoxia-inducible factor-2α)-regulated genes, and tobacco smoke decreases pulmonary HIF-2α concentrations. These findings suggest that decreased HIF-2α expression is important in the development of emphysema.Objectives: The objective of this study was to evaluate the roles of endothelial-cell (EC) HIF-2α in the pathogenesis of emphysema in mice.Methods: Mouse lungs were examined for emphysema after either the loss or the overexpression of EC Hif-2α. In addition, SU5416, a VEGFR2 inhibitor, was used to induce emphysema. Lungs were evaluated for HGF (hepatocyte growth factor), a protein involved in alveolar development and homeostasis. Lungs from patients with emphysema were measured for endothelial HIF-2α expression.Measurements and Main Results: EC Hif-2α deletion resulted in emphysema in association with fewer ECs and pericytes. After SU5416 exposure, EC Hif-2α-knockout mice developed more severe emphysema, whereas EC Hif-2α-overexpressing mice were protected. EC Hif-2α-knockout mice demonstrated lower levels of HGF. Human emphysema lung samples exhibited reduced EC HIF-2α expression.Conclusions: Here, we demonstrate a unique protective role for pulmonary endothelial HIF-2α and how decreased expression of this endogenous factor causes emphysema; its pivotal protective function is suggested by its ability to overcome VEGF antagonism. HIF-2α may maintain alveolar architecture by promoting vascular survival and associated HGF production. In summary, HIF-2α may be a key endogenous factor that prevents the development of emphysema, and its upregulation has the potential to foster lung health in at-risk patients.

IL‐17 Signaling Mediates Cytolytic Natural Killer Cell Activation in Placental‐Ischemic Rats

The Reduced Uterine Perfusion Pressure (RUPP) rat model of placental ischemia mimics many characteristics of preeclampsia (PE) including hypertension (HTN), fetal growth restriction, and increased TH17s, IL‐17, and cytolytic natural killer cells (cNKs). IL‐17 stimulates NK cytotoxicity in vitro. This study tested the hypothesis that blockade of IL‐17 signaling would inhibit activation of cNKs to improve blood pressure and fetal growth in RUPP rats. On gestation day (GD) 14, osmotic pumps infusing IL‐17 RC (100 pg/d), a soluble receptor for IL‐17, were implanted into pregnant rats undergoing RUPP (RUPP+IL17 RC). On GD 19, circulating and placental cNKs were quantified via flow cytometry in normal pregnant (NP, n=9), RUPP (n=9), and RUPP+IL17 RC (n=9) rats. cNK activation, placental ROS, fetal and placental weights, circulating and placental cytokines, and MAP were also assessed. Placental cNKs (% gated), increased from 7.2±2.8% in NP to 16.6±3.3% in RUPP (p&lt;0.05 vs NP), and were normalized to 6.8±1.7% after IL‐17 blockade (p&lt;0.05 vs RUPP). Similarly, circulating cNKs increased from 6.1±1.4% in NP to 16.7±3.2% in RUPP (p&lt;0.05 vs NP) and decreased to 8.1±1.9% in RUPP+IL‐17 RC (p&lt;0.05 vs RUPP). Placental levels of the cNK cytokine, TNFα, increased from 25.9±3.0 pg/mg in NP to 44.8±5.0 pg/mg in RUPP (p&lt;0.05 vs NP) and was normalized to 28.5±4.6 pg/mg in RUPP+IL‐17 RC (p&lt;0.05 vs RUPP). Similarly, placental levels of MIP3a, a cNK chemokine, increased from 0.84±0.1 pg/mg in NP to 1.4±0.2 pg/mg in RUPP (p&lt;0.05 vs NP) and decreased to 0.76±0.1 pg/mg in RUPP+IL‐17 RC (p&lt;0.05 vs RUPP). Assessment of in vitro cytotoxic activity of placental NKs from rats in each group demonstrated a 5‐fold increase in cytotoxicity by RUPP NKs (n=6) compared to NP NKs (n=6) (p&lt;0.05 vs NP). Cytotoxicity of NKs from RUPP+IL‐17 RC rats (n=6) was not significantly higher than NP NKs (3‐fold higher, p&gt;0.05 vs NP or RUPP). Circulating levels of VEGF decreased from 163±11 pg/mL in NP to 88±16 pg/mL RUPP (p&lt;0.05 vs NP) and increased to 195±12 pg/mL in RUPP+IL‐17 RC (p&lt;0.05 vs RUPP). Soluble fms‐like tyrosine kinase 1 (sFlt‐1), a VEGF antagonist, increased from 196±100 pg/mL in NP to 963±114 pg/mL in RUPP (p&lt;0.05 vs NP) and decreased to 249±78 pg/mL in RUPP+IL‐17 RC (p&lt;0.05 vs RUPP). Placental ROS (RLU/min/mg) was increased from 890±218 in NP (n=6) to 2240±319 in RUPP (n=6, p&lt;0.05 vs NP) and decreased to 1145±137 with IL‐17 blockade (n=5, p&lt;0.05 vs RUPP). Fetal weight decreased from 2.4±0.04 g in NP to 2.0±0.06 g in RUPP (p&lt;0.05 vs NP) and increased to 2.2±0.04 g in RUPP+IL17 RC (p&lt;0.05 vs RUPP). Placental weights followed a similar trend. MAP increased from 90±2 mmHg in NP to 122±3 mmHg in RUPP (p&lt;0.05 vs NP), and was decreased to 103±3 mmHg in RUPP+IL17 RC (p&lt;0.05 vs RUPP, p&lt;0.05 vs NP). These data demonstrate a direct role for IL‐17 signaling to mediate cNK activation in response to placental ischemia during pregnancy. By better understanding this stimulus, novel therapeutics can be developed to inhibit cytolytic NK activation while also retaining the critical roles of normal NKs in pregnancy maintenance and immune defense. Targeting IL‐17 signaling may be effective in ameliorating several pathophysiologies of PE and could improve both maternal and fetal outcomes.Support or Funding InformationR00HL130456, T32HL105324

Intraocular VEGF deprivation induces degeneration and fibrogenic response in retina.

VEGF is a critical driver of ocular neovascularization under disease conditions. Current therapeutic strategies rely on intraocular delivery of VEGF-antagonizing reagents, which results in sustained suppression of pathogenic vascularization. Although significant advancement has been achieved in VEGF antagonism, substantial adverse effects have been reported in retrospective clinical studies. To study mechanisms for VEGF antagonism-associated adverse effects in visual system, we intravitreally delivered recombinant adeno-associated virus-mediated expression of soluble Fms-related tyrosine kinase-1 (rAAV.sFLT-1), the extracellular domain of VEGF receptor, and analyzed the morphology and functions of retinal tissue. Here, we confirmed that intraocular VEGF antagonism induced retinal degeneration and gliosis. The functional deficit in retinal response to visual stimulation was also demonstrated in rAAV.sFLT-1-treated eyes by electroretinogram. Moreover, high-throughput RNA sequencing analysis suggests that VEGF antagonism activates retinal degeneration, inflammation, and other adverse effects. Taken together, our findings have shed light on pathogenic mechanisms for VEGF antagonism-associated adverse effects and potential therapeutic targets.-Xiao, M., Liu, Y., Wang, L., Liang, J., Wang, T., Zhai, Y., Wang, Y., Liu, S., Liu, W., Luo, X., Wang, F., Sun, X. Intraocular VEGF deprivation induces degeneration and fibrogenic response in retina.

Therapeutic Effect of Garcinia cambogia Extract and Hydroxycitric Acid Inhibiting Hypoxia-Inducible Factor in a Murine Model of Age-Related Macular Degeneration.

Background: Age-related macular degeneration (AMD) is the leading cause of blindness and can be classified into two types called atrophic AMD (dry AMD) and neovascular AMD (wet AMD). Dry AMD is characterized by cellular degeneration of the retinal pigment epithelium, choriocapillaris, and photoreceptors. Wet AMD is characterized by the invasion of abnormal vessels from the choroid. Although anti-vascular endothelial growth factor (VEGF) therapy has a potent therapeutic effect against the disease, there is a possibility of chorio-retinal atrophy and adverse systemic events due to long-term robust VEGF antagonism. We focused on hypoxia-inducible factor (HIF) regulation of VEGF transcription, and report the suppressive effects of HIF inhibition against ocular phenotypes in animal models. Many of the known HIF inhibitors are categorized as anti-cancer drugs, and their systemic side effects are cause for concern in clinical use. In this study, we explored food ingredients that have HIF inhibitory effects and verified their effects in an animal model of AMD. Methods: Food ingredients were screened using a luciferase assay. C57BL6/J mice were administered the Garcinia cambogia extract (Garcinia extract) and hydroxycitric acid (HCA). Choroidal neovascularization (CNV) was induced by laser irradiation. Results: Garcinia extract and HCA showed inhibitory effects on HIF in the luciferase assay. The laser CNV model mice showed significant reduction of CNV volume by administering Garcinia extract and HCA. Conclusions: Garcinia extract and HCA showed therapeutic effects in a murine AMD model.

Fluocinolone acetonide 0.19 mg intravitreal implant improves foveal thickness and reduces treatment burden for up to 1 year in eyes with persistent diabetic macular edema.

Objective: To assess the effectiveness, safety, and treatment burden in eyes with persistent diabetic macular edema (DME) for up to 1 year after administration of 0.19 mg fluocinolone acetonide (FAc) implant (Iluvien®).Methods: This retrospective study at one private practice in the US included 40 eyes from 33 patients treated with an FAc implant. Eyes had previously been treated with VEGF antagonists, dexamethasone, or focal laser. The primary outcome was change in central foveal thickness from baseline. Data were also collected on demographics, visual acuity, intraocular pressure (IOP), use of IOP-lowering drugs for elevated IOP, lens clarity, and treatment burden before and after the implant.Results: Average duration of diabetes and DME at baseline was 19 and 5 years, respectively, and average glycated hemoglobin was 7.21%. Severity of diabetic retinopathy before the implant had a slight bimodal distribution: moderate nonproliferative diabetic retinopathy (35%) and proliferative diabetic retinopathy (40%). Improvements in central foveal thickness from baseline were evident at 3 months (mean change –74.2 µm, P<0.001) and sustained through 12 months (–55.3 µm; P=0.005) for most eyes. Mean visual acuity remained stable (66.2 letters at baseline versus 67.2 letters at 12 months, roughly equivalent to 20/50 vision; P=0.855). On average, eyes required one anti-VEGF injection every 1.9 months before and one every 6.6 months after the implant, while 60% of eyes did not require additional anti-VEGF injections. Small but significant increases in IOP at months 3, 6, and 9 were not sustained to month 12, and mean IOP was normal throughout follow-up.Conclusion: In patients with DME previously treated with a steroid, and treated according to licensed indications in the US, an FAc implant not only reduces the burden of disease in the real-world setting, but also the burden of injections and office visits for patients.

Pharmacological HIF inhibition prevents retinal neovascularization with improved visual function in a murine oxygen-induced retinopathy model

Neovascular retinal diseases are the leading causes of blindness in advanced countries. To date, anti-VEGF (vascular endothelial growth factor) drugs are clinically effective and widely used for these diseases. However, recent animal and clinical studies reported that potent and long-term VEGF antagonism may induce chorioretinal atrophy. Thus, physiological amount of VEGF is required for the homeostasis in the retina. Hypoxia-inducible factors (HIFs) are transcription factors located upstream of VEGF. We hypothesized that ectopically stabilized HIFs induce pathological amount of VEGF involved with retinal neovascularization. Therefore, HIF inhibition could be an alternative therapeutic candidate targeting the pathological amount of VEGF while holding a physiological amount of VEGF. To test this hypothesis, topotecan and doxorubicin, HIF inhibitors with different mechanisms were administered to the murine oxygen-induced retinopathy (OIR) model. We found that both topotecan and doxorubicin significantly prevented pathological but not physiological neovascularization in OIR. Furthermore, impaired visual function observed in OIR can also be suppressed by administering topotecan. These data suggested that HIF inhibition may be effective for pathological angiogenesis and neurodegeneration of the retina.

Clinical study of laser photocoagulation combined with VEGF antagonists for DME

Clinical study of laser photocoagulation combined with VEGF antagonists for DME