Therapeutic angiogenesis is an experimental frontier in vascular biology that seeks to deliver angiogenic growth factors to ischemic or injured tissues to promote targeted formation of new blood vessels as an alternative approach to surgical revascularization procedures. Vascular endothelial growth factor (VEGF) is a potent angiogenic signal protein that is locally upregulated at sites of tissue injury. However, therapies aimed at increasing VEGF levels experimentally by injecting VEGF gene or protein failed to improve outcomes in human trials in part due to its short half-life and systemic toxicity. We recently designed a novel 12-amino acid peptide (PR1P) whose sequence was derived from an extracellular VEGF-binding domain of the pro-angiogenic glycoprotein prominin-1. In this study, we characterized the molecular binding properties of this novel potential therapeutic for targeted angiogenesis and provided the foundation for its use as an angiogenic molecule that can potentiate endogenous VEGF. We showed that PR1P bound VEGF directly and enhanced VEGF binding to endothelial cells and to VEGF receptors VEGFR2 and neuropilin-1. PR1P increased angiogenesis in the murine corneal micropocket assay when combined with VEGF, but had no activity without added VEGF. In addition, PR1P also enhanced angiogenesis in murine choroidal neovascularization and wound-healing models and augmented reperfusion in a murine hind-limb ischemia model. Together our data suggest that PR1P enhanced angiogenesis by potentiating the activity of endogenous VEGF. In so doing, this novel therapy takes advantage of endogenous VEGF gradients generated in injured tissues and may improve the efficacy of and avoid systemic toxicity seen with previous VEGF therapies.
Angiogenesis plays a key role in the pathology of diseases such as cancer, diabetic retinopathy, and age-related macular degeneration. Understanding the driving forces of endothelial cell migration and organization, as well as the time frame of these processes, can elucidate mechanisms of action of important pathological pathways. Herein, we have developed an organ-specific microfluidic platform recapitulating the in vivo angiogenic microenvironment by co-culturing mouse primary brain endothelial cells with brain pericytes in a three-dimensional (3D) collagen scaffold. As a proof of concept, we show that this model can be used for studying the angiogenic process and further comparing the angiogenic properties between two different common inbred mouse strains, C57BL/6J and 129S1/SvlmJ. We further show that the newly discovered angiogenesis-regulating gene Padi2 promotes angiogenesis through Dll4/Notch1 signaling by an on-chip mechanistic study. Analysis of the interplay between primary endothelial cells and pericytes in a 3D microfluidic environment assists in the elucidation of the angiogenic response.
Blood leakage from the vessels in the eye is the hallmark of many vascular eye diseases. One of the preclinical mouse models of retinal blood leakage, the very-low-density-lipoprotein receptor deficient mouse (Vldlr-/-), is used for drug screening and mechanistic studies. Vessel leakage is usually examined using Fundus fluorescein angiography (FFA). However, interpreting FFA images of the Vldlr-/- model is challenging as no automated and objective techniques exist for this model. A pipeline has been developed for quantifying leakage intensity and area including three tasks: (i) blood leakage identification, (ii) blood vessel segmentation, and (iii) image registration. Morphological operations followed by log-Gabor quadrature filters were used to identify leakage regions. In addition, a novel optic disk detection algorithm based on graph analysis was developed for registering the images at different timepoints. Blood leakage intensity and area measured by the methodology were compared to ground truth quantifications produced by two annotators. The relative difference between the quantifications from the method and those obtained from ground truth images was around 10% ± 6% for leakage intensity and 17% ± 8% for leakage region. The Pearson correlation coefficient between the method results and the ground truth was around 0.98 for leakage intensity and 0.94 for leakage region. Therefore, we presented a computational method for quantifying retinal vascular leakage and vessels using FFA in a preclinical angiogenesis model, the Vldlr-/- model.
Vascular endothelial growth factor (VEGF) plays a crucial role in developmental and pathological angiogenesis. Expression of VEGF in quiescent adult tissue suggests a potential role in the maintenance of mature blood vessels. We demonstrate, using a Vegf-lacZ reporter mouse model, that VEGF is expressed by arterial but not by venous or capillary endothelial cells (ECs) in vivo. Using an in vitro model, we show that arterial shear stress of human umbilical vein ECs (HUVECs) decreases apoptosis and increases VEGF expression, which is mediated by the induction of Krüppel-like factor 2 (KLF2). Additionally, shear stress stimulates the expression of VEGF receptor 2 (VEGFR2) and is associated with its activation. Knockdown of VEGF in shear stressed HUVECs blocks the protective effect of shear stress, resulting in EC apoptosis equivalent to that in control ECs cultured under static conditions. Similarly, treatment of ECs subjected to arterial shear stress with the VEGF receptor tyrosine kinase inhibitor SU1498, or VEGFR2 neutralizing antiserum, led to increased apoptosis, demonstrating that the mechanoprotection from increased shear is mediated by VEGFR2. Taken together, these studies suggest that arterial flow induces VEGF-VEGFR2 autocrine-juxtacrine signaling, which is a previously unidentified mechanism for vascular EC survival in adult arterial blood vessels.
The cornea actively maintains its own avascular status to preserve its ultimate optical function. This corneal avascular state is also defined as "corneal angiogenic privilege", which results from a critical and sensitive balance between anti-angiogenic and pro-angiogenic mechanisms. In our review, we aim to explore the complex equilibrium among multiple mediators which prevents neovascularization in the resting cornea, as well as to unveil the evolutive process which leads to corneal angiogenesis in response to different injuries.
Angiogenesis, disruption of the retinal barrier, leukocyte-adhesion and oedema are cardinal signs of proliferative retinopathies that are associated with vision loss. Therefore, identifying factors that regulate these vascular dysfunctions is critical to target pathological angiogenesis. Given the conflicting role of bioactive lipids reported in the current literature, the goal of this review is to provide the reader a clear road map of what has been accomplished so far in the field with specific focus on the role of polyunsaturated fatty acids (PUFAs)-derived metabolites in proliferative retinopathies. This necessarily entails a description of the different retina cells, blood retina barriers and the role of (PUFAs)-derived metabolites in diabetic retinopathy, retinopathy of prematurity and age-related macular degeneration as the most common types of proliferative retinopathies.
Pathologic angiogenesis causes blindness in many eye diseases. Crespo-Garcia, Tsuruda, and Dejda et al. employed bioinformatics to characterize cell senescence as a primary factor in the common pathogenesis of retinopathies. They validated their findings using human and mouse retina with proliferative retinopathy. Clearance of senescent cells suppressed neovessel growth.
Higgs C, Crow YJ, Adams DM, Chang E, Hayes D, Herbig U, Huang JN, Himes R, Jajoo K, Johnson BF, Reynolds SD, Yonekawa Y, Armanios M, Boulad F, DiNardo CD, Dufour C, Goldman FD, Khan S, Kratz C, Myers KC, Raghu G, Alter BP, Aubert G, Bhala S, Cowen EW, Dror Y, El-Youssef M, Friedman B, Giri N, Helms Guba L, Khincha PP, Lin TF, Longhurst H, McReynolds LJ, Nelson A, Olson T, Pariser A, Perona R, Sasa G, Schratz K, Simonetto DA, Townsley D, Walsh M, Stevens K, Agarwal S, Bertuch AA, Savage SA, for (CCCTAA) CCCT-associated A. Understanding the evolving phenotype of vascular complications in telomere biology disorders. Angiogenesis 2019;22(1):95-102.Abstract
Vascular complications such as bleeding due to gastrointestinal telangiectatic anomalies, pulmonary arteriovenous malformations, hepatopulmonary syndrome, and retinal vessel abnormalities are being reported in patients with telomere biology disorders (TBDs) more frequently than previously described. The international clinical care consortium of telomere-associated ailments and family support group Dyskeratosis Congenita Outreach, Inc. held a workshop on vascular abnormalities in the TBDs at the National Cancer Institute in October 2017. Clinicians and basic scientists reviewed current data on vascular complications, hypotheses for the underlying biology and developed new collaborations to address the etiology and clinical management of vascular complications in TBDs.
The role of apoptosis in the formation and regression of neovascularization is largely hypothesized, although the detailed mechanism remains unclear. Inflammatory cells and endothelial cells both participate and interact during neovascularization. During the early stage, these cells may migrate into an angiogenic site and form a pro-angiogenic microenvironment. Some angiogenic vessels appear to regress, whereas some vessels mature and remain. The control mechanisms of these processes, however, remain unknown. Previously, we reported that the prevention of mitochondrial apoptosis contributed to cellular survival via the prevention of the release of proapoptotic factors, such as apoptosis-inducing factor (AIF) and cytochrome c. In this study, we investigated the regulatory role of cellular apoptosis in angiogenesis using two models of ocular neovascularization: laser injury choroidal neovascularization and VEGF-induced corneal neovascularization in AIF-deficient mice. Averting apoptosis in AIF-deficient mice decreased apoptosis of leukocytes and endothelial cells compared to wild-type mice and resulted in the persistence of these cells at angiogenic sites in vitro and in vivo. Consequently, AIF deficiency expanded neovascularization and diminished vessel regression in these two models. We also observed that peritoneal macrophages from AIF-deficient mice showed anti-apoptotic survival compared to wild-type mice under conditions of starvation. Our data suggest that AIF-related apoptosis plays an important role in neovascularization and that mitochondria-regulated apoptosis could offer a new target for the treatment of pathological angiogenesis.
Angiogenesis, in which vascular endothelial growth factor receptor (VEGFR) 2 plays an essential role, is associated with a variety of human diseases including proliferative diabetic retinopathy and wet age-related macular degeneration. Here we report that a system of adeno-associated virus (AAV)-mediated clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas)9 from Streptococcus pyogenes (SpCas9) is used to deplete VEGFR2 in vascular endothelial cells (ECs), whereby the expression of SpCas9 is driven by an endothelial-specific promoter of intercellular adhesion molecule 2. We further show that recombinant AAV serotype 1 (rAAV1) transduces ECs of pathologic vessels, and that editing of genomic VEGFR2 locus using rAAV1-mediated CRISPR/Cas9 abrogates angiogenesis in the mouse models of oxygen-induced retinopathy and laser-induced choroid neovascularization. This work establishes a strong foundation for genome editing as a strategy to treat angiogenesis-associated diseases.Abnormal angiogenesis causes many ocular diseases. Here the authors employ CRISPR/Cas9 gene editing technology to silence VEGFR2, a major regulator of angiogenesis, in retinal endothelium and abrogate angiogenesis in the mouse models of oxygen-induced retinopathy and laser-induced choroid neovascularization.
Recent findings indicate that growth factor-driven angiogenesis is markedly influenced by genetic variation. This variation in angiogenic responsiveness may alter the susceptibility to a number of angiogenesis-dependent diseases. Here, we utilized the genetic diversity available in common inbred mouse strains to identify the loci and candidate genes responsible for differences in angiogenic response. The corneal micropocket neovascularization assay was performed on 42 different inbred mouse strains using basic fibroblast growth factor (bFGF) pellets. We performed a genome-wide association study utilizing efficient mixed-model association (EMMA) mapping using the induced vessel area from all strains. Our analysis yielded five loci with genome-wide significance on chromosomes 4, 8, 11, 15 and 16. We further refined the mapping on chromosome 4 within a haplotype block containing multiple candidate genes. These genes were evaluated by expression analysis in corneas of various inbred strains and in vitro functional assays in human microvascular endothelial cells (HMVECs). Of these, we found the expression of peptidyl arginine deiminase type II (Padi2), known to be involved in metabolic pathways, to have a strong correlation with a haplotype shared by multiple high angiogenic strains. In addition, inhibition of Padi2 demonstrated a dosage-dependent effect in HMVECs. To investigate its role in vivo, we knocked down Padi2 in transgenic kdrl:zsGreen zebrafish embryos using morpholinos. These embryos had disrupted vessel formation compared to control siblings. The impaired vascular pattern was partially rescued by human PADI2 mRNA, providing evidence for the specificity of the morphant phenotype. Taken together, our study is the first to indicate the potential role of Padi2 as an angiogenesis-regulating gene. The characterization of Padi2 and other genes in associated pathways may provide new understanding of angiogenesis regulation and novel targets for diagnosis and treatment of a wide variety of angiogenesis-dependent diseases.
We have previously used the genetic diversity available in common inbred mouse strains to identify quantitative trait loci (QTLs) responsible for the differences in angiogenic response using the corneal micropocket neovascularization (CoNV) assay. Employing a mouse genome-wide association study (GWAS) approach, the region on chromosome 15 containing Basp1 was identified as being significantly associated with angiogenesis in inbred strains. Here, we developed a unique strategy to determine and verify the role of BASP1 in angiogenic pathways. Basp1 expression in cornea had a strong correlation with a haplotype shared by mouse strains with varied angiogenic phenotypes. In addition, inhibition of BASP1 demonstrated a dosage-dependent effect in both primary mouse brain endothelial and human microvascular endothelial cell (HMVEC) migration. To investigate its role in vivo, we knocked out basp1 in transgenic kdrl:zsGreen zebrafish embryos using a widely adopted CRISPR-Cas9 system. These embryos had severely disrupted vessel formation compared to control siblings. We further show that basp1 promotes angiogenesis by upregulating β-catenin gene and the Dll4/Notch1 signaling pathway. These results, to the best of our knowledge, provide the first in vivo evidence to indicate the role of Basp1 as an angiogenesis-regulating gene and opens the potential therapeutic avenues for a wide variety of systemic angiogenesis-dependent diseases.
In 1994, The American Journal of Pathology published a key article reporting that hypoxic retina produces vascular endothelial growth factor (VEGF), suggesting a role for VEGF in ocular neovascularization. Subsequent developments in anti-VEGF treatment for neovascular eye disease have improved visual outcomes and changed the standard of care in retinal medicine and ophthalmology.
Verteporfin (VP) was first used in Photodynamic therapy, where a non-thermal laser light (689 nm) in the presence of oxygen activates the drug to produce highly reactive oxygen radicals, resulting in local cell and tissue damage. However, it has also been shown that Verteporfin can have non-photoactivated effects such as interference with the YAP-TEAD complex of the HIPPO pathway, resulting in growth inhibition of several neoplasias. More recently, it was proposed that, another non-light mediated effect of VP is the formation of cross-linked oligomers and high molecular weight protein complexes (HMWC) that are hypothesized to interfere with autophagy and cell growth. Here, in a series of experiments, using human uveal melanoma cells (MEL 270), human embryonic kidney cells (HEK) and breast cancer cells (MCF7) we showed that Verteporfin-induced HMWC require the presence of light. Furthermore, we showed that the mechanism of this cross-linking, which involves both singlet oxygen and radical generation, can occur very efficiently even after lysis of the cells, if the lysate is not protected from ambient light. This work offers a better understanding regarding VP's mechanisms of action and suggests caution when one studies the non-light mediated actions of this drug.
Proliferative diabetic retinopathy (PDR) is a common cause of blindness in the developed world's working adult population and affects those with type 1 and type 2 diabetes. We identified Runt-related transcription factor 1 (RUNX1) as a gene upregulated in CD31(+) vascular endothelial cells obtained from human PDR fibrovascular membranes (FVMs) via transcriptomic analysis. In vitro studies using human retinal microvascular endothelial cells (HRMECs) showed increased RUNX1 RNA and protein expression in response to high glucose, whereas RUNX1 inhibition reduced HRMEC migration, proliferation, and tube formation. Immunohistochemical staining for RUNX1 showed reactivity in vessels of patient-derived FVMs and angiogenic tufts in the retina of mice with oxygen-induced retinopathy, suggesting that RUNX1 upregulation is a hallmark of aberrant retinal angiogenesis. Inhibition of RUNX1 activity with the Ro5-3335 small molecule resulted in a significant reduction of neovascular tufts in oxygen-induced retinopathy, supporting the feasibility of targeting RUNX1 in aberrant retinal angiogenesis.
We have previously shown that knockdown of endomucin (EMCN), an integral membrane glycocalyx glycoprotein, prevents VEGF-induced proliferation, migration, and tube formation and angiogenesis . In the endothelium, VEGF mediates most of its angiogenic effects through VEGF receptor 2 (VEGFR2). To understand the role of EMCN, we examined the effect of EMCN depletion on VEGFR2 endocytosis and activation. Results showed that although VEGF stimulation promoted VEGFR2 internalization in control endothelial cells (ECs), loss of EMCN prevented VEGFR2 endocytosis. Cell surface analysis revealed a decrease in VEGFR2 following VEGF stimulation in control but not siRNA directed against EMCN-transfected ECs. EMCN depletion resulted in heightened phosphorylation following VEGF stimulation with an increase in total VEGFR2 protein. These results indicate that EMCN modulates VEGFR2 endocytosis and activity and point to EMCN as a potential therapeutic target.-LeBlanc, M. E., Saez-Torres, K. L., Cano, I., Hu, Z., Saint-Geniez, M., Ng, Y.-S., D'Amore, P. A. Glycocalyx regulation of vascular endothelial growth factor receptor 2 activity.
Pathological angiogenesis in the eye is an important feature in the pathophysiology of many vision-threatening diseases, including retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration, as well as corneal diseases with abnormal angiogenesis. Development of reproducible and reliable animal models of ocular angiogenesis has advanced our understanding of both the normal development and the pathobiology of ocular neovascularization. These models have also proven to be valuable experimental tools with which to easily evaluate potential antiangiogenic therapies beyond eye research. This review summarizes the current available animal models of ocular angiogenesis. Models of retinal and choroidal angiogenesis, including oxygen-induced retinopathy, laser-induced choroidal neovascularization, and transgenic mouse models with deficient or spontaneous retinal/choroidal neovascularization, as well as models with induced corneal angiogenesis, are widely used to investigate the molecular and cellular basis of angiogenic mechanisms. Theoretical concepts and experimental protocols of these models are outlined, as well as their advantages and potential limitations, which may help researchers choose the most suitable models for their investigative work.-Liu, C.-H., Wang, Z., Sun, Y., Chen, J. Animal models of ocular angiogenesis: from development to pathologies.
Pathological angiogenesis is a hallmark of various vascular diseases, including vascular eye disorders. Dysregulation of microRNAs (miRNAs), a group of small regulatory RNAs, has been implicated in the regulation of ocular neovascularization. This study investigated the specific role of microRNA-145 (miR-145) in regulating vascular endothelial cell (EC) function and pathological ocular angiogenesis in a mouse model of oxygen-induced retinopathy (OIR). Expression of miR-145 was significantly upregulated in OIR mouse retinas compared with room air controls. Treatment with synthetic miR-145 inhibitors drastically decreased levels of pathological neovascularization in OIR, without substantially affecting normal developmental angiogenesis. In cultured human retinal ECs, treatment with miR-145 mimics significantly increased the EC angiogenic function, including proliferation, migration, and tubular formation, whereas miR-145 inhibitors attenuated in vitro angiogenesis. Tropomodulin3 (TMOD3), an actin-capping protein, is a direct miR-145 target and is downregulated in OIR retinas. Treatment with miR-145 mimic led to TMOD3 inhibition, altered actin cytoskeletal architecture, and elongation of ECs. Moreover, inhibition of TMOD3 promoted EC angiogenic function and pathological neovascularization in OIR and abolished the vascular effects of miR-145 inhibitors in vitro and in vivo. Overall, our findings indicate that miR-145 is a novel regulator of TMOD3-dependent cytoskeletal architecture and pathological angiogenesis and a potential target for development of treatments for neovascular eye disorders.
The coordination of metabolic signals among different cellular components in pathological retinal angiogenesis is poorly understood. Here, we showed that in the pathological angiogenic vascular niche, retinal myeloid cells, particularly macrophages/microglia that are spatially adjacent to endothelial cells (ECs), are highly glycolytic. We refer to these macrophages/microglia that exhibit a unique angiogenic phenotype with increased expression of both M1 and M2 markers and enhanced production of both proinflammatory and proangiogenic cytokines as pathological retinal angiogenesis-associated glycolytic macrophages/microglia (PRAGMs). The phenotype of PRAGMs was recapitulated in bone marrow-derived macrophages or retinal microglia stimulated by lactate that was produced by hypoxic retinal ECs. Knockout of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (; for rodents), a glycolytic activator in myeloid cells, impaired the ability of macrophages/microglia to acquire an angiogenic phenotype, rendering them unable to promote EC proliferation and sprouting and pathological neovascularization in a mouse model of oxygen-induced proliferative retinopathy. Mechanistically, hyperglycolytic macrophages/microglia produced large amount of acetyl-coenzyme A, leading to histone acetylation and PRAGM-related gene induction, thus reprogramming macrophages/microglia into an angiogenic phenotype. These findings reveal a critical role of glycolytic metabolites as initiators of reciprocal activation of macrophages/microglia and ECs in the retinal angiogenic niche and suggest that strategies targeting the metabolic communication between these cell types may be efficacious in the treatment of pathological retinal angiogenesis.