Abnormal Blood Vessel Growth Research Articles

Gamma-Secretase Inhibitor Treatment Promotes VEGF-A-Driven Blood Vessel Growth and Vascular Leakage but Disrupts Neovascular Perfusion

The Notch signaling pathway is essential for normal development due to its role in control of cell differentiation, proliferation and survival. It is also critically involved in tumorigenesis and cancer progression. A key enzyme in the activation of Notch signaling is the gamma-secretase protein complex and therefore, gamma-secretase inhibitors (GSIs)—originally developed for Alzheimer's disease—are now being evaluated in clinical trials for human malignancies. It is also clear that Notch plays an important role in angiogenesis driven by Vascular Endothelial Growth Factor A (VEGF-A)—a process instrumental for tumor growth and metastasis. The effect of GSIs on tumor vasculature has not been conclusively determined. Here we report that Compound X (CX), a GSI previously reported to potently inhibit Notch signaling in vitro and in vivo, promotes angiogenic sprouting in vitro and during developmental angiogenesis in mice. Furthermore, CX treatment suppresses tumor growth in a mouse model of renal carcinoma, leads to the formation of abnormal vessels and an increased tumor vascular density. Using a rabbit model of VEGF-A-driven angiogenesis in skeletal muscle, we demonstrate that CX treatment promotes abnormal blood vessel growth characterized by vessel occlusion, disrupted blood flow, and increased vascular leakage. Based on these findings, we propose a model for how GSIs and other Notch inhibitors disrupt tumor blood vessel perfusion, which might be useful for understanding this new class of anti-cancer agents.

Retinopathy of prematurity: understanding ischemic retinal vasculopathies at an extreme of life

Retinopathy of prematurity (ROP) is a major complication of preterm birth. It encompasses a spectrum of pathologies that affect vision, from mild disease that resolves spontaneously to severe disease that causes retinal detachment and subsequent blindness. The pathologies are characterized by an arrest in normal retinal vascular development associated with microvascular degeneration. The resulting ischemia and retinal hypoxia lead to excessive abnormal compensatory blood vessel growth. However, this neovascularization can lead to fibrous scar formation and culminate in retinal detachment. Present therapeutic modalities to limit the adverse consequences of aberrant neovascularization are invasive and/or tissue-destructive. In this Review, we discuss current concepts on retinal microvascular degeneration, neovascularization, and available treatments, as well as present future perspectives toward more profound elucidation of the pathogenesis of ROP.

TRANSLATIONAL IMPACT

von Hippel-Lindau disease (VHL) is a rare, genetic multisystem disorder characterized by the abnormal growth of blood vessels and tumors. Between 60% and 80% of all patients have benign hemangioblastomas of the retina and central nervous system (CNS) that frequently cause neurological morbidity and mortality. VHL patients can also develop retinal neovascularization (also termed angiogenesis; the formation of new blood vessels), similar to diabetic retinopathy and age-related macular degeneration. Angiogenic retinopathies exhibit vascular leakage, macular edema (fluid accumulation) and detachment of the retina, and are the biggest cause of blindness in the Western world, with very limited treatment options. Unfortunately, the current models for angiogenesis do not fully recapitulate the complexity of angiogenic-related disorders. For instance, hemangioblastomas of the retina and CNS have not been found in any of the VHL mouse models.The VHL tumor suppressor gene, VHL, is crucial for oxygen homeostasis. Its loss causes upregulation of HIF (hypoxia-inducible factor), a transcription factor with a central role in oxygen-regulated gene expression, which in turn increases the expression of hypoxia-inducible mRNAs, including those encoding vascular endothelial growth factor A (VEGFA), VEGFA receptors, and proteins involved in the HIF signaling pathwayIn this study, the authors show that zebrafish vhl mutants provide an in vivo model for pathological angiogenesis and vascular retinopathies. Using blood vessel-specific transgenes, they show that vhl mutants develop severe neovascularization in the brain, eye and trunk. The retinal vasculature of vhl−/− zebrafish is leaky, with severe macular edema and retinal detachment, mimicking human angiogenic retinopathies. As in humans, the zebrafish VEGFA orthologs, Vegfaa/Vegfab and Vegfb, are upregulated by loss of vhl, and are key effectors of the vhl−/− angiogenic phenotype; treatment with VEGF receptor inhibitors can block pathological angiogenesis in mutant fish.These zebrafish vhl mutants can be used to understand the mechanisms underlying the development of vascular lesions in VHL disease, and are a unique and clinically relevant angiogenesis model, as pathological angiogenesis and vascular retinopathies can be studied non-invasively under normoxic conditions. Zebrafish vhl mutant embryos with fluorescently-labeled blood vessels are also a cost-effective and efficient model for whole organism screening of anti-angiogenic pharmacological agents and combinatorial therapies.

Frontiers in Microcirculation: Control Processes and Clinical Applications

This edition of Microcirculation presents five current and emerging perspectives of the microcirculation in development, health, and disease. The onset of blood flow and pressure are central to cardiovascular development. These hemodynamic forces are explored in light of underlying molecular signaling pathways that affect vascular and cardiac cell shape and proliferation. Shear-induced strain exerted on the plasma membrane and cytoskeleton is transmitted to cell nuclei and thereby affects gene activation through mechanotransduction. Altered stiffness or disturbed surfaces of aberrant vascular cells may affect an array of vasculopathies through altered gene expression. The endothelium is both a site and source for inflammatory responses triggered by cardiovascular risk factors often mediated through reactive oxygen species and angiotensin II. Tumor microenvironments are disturbed with abnormal growth and remodeling of blood and lymphatic vessels. More effective targeting strategies for delivering anti-angiogenic and cytotoxic agents are being developed through advances in intravital imaging. Blood flow control requires both vasodilation and vasoconstriction to be coordinated along and among arterioles and feed arteries. Evolving insights into signaling pathways between smooth muscle cells and endothelial cells illuminate how such processes can be affected in vasculopathies. These timely reviews provide a novel reference for advancing research frontiers in microcirculation.

Neovascularization in diabetes

Diabetes and its complications are a major public health burden in the developed world. The major cause of diabetic complications is abnormal growth of new blood vessels. This dysfunctional neovascularization results in significant morbidity and mortality in patients with diabetes and, as such, is a major focus of basic and clinical investigation. It has become clear that hyperglycemia disrupts tissue-level signaling in response to hypoxia and ischemia, impairs the vasculogenic potential of circulating stem cells and fundamentally alters the structure and function of key neovascularization proteins, including hypoxia-inducible factor-1. These mechanistic and pathophysiologic studies have revealed new therapeutic targets to restore normal neovascularization and to ameliorate and prevent diabetic vascular complications.

Retinopathy of Prematurity: An Eye Toward Better Outcomes

Retinopathy of prematurity (ROP) results from the abnormal growth of blood vessels in the vascular bed supporting the developing retina. Estimated to cause up to 500 new cases of blindness in the U.S. each year, ROP affects primarily infants born at less than 1,500 g. Although its etiology is not well understood, ROP is thought to occur as a result of a complex interaction between oxygen and vascular growth factors. This article briefly reviews the history of ROP, discusses its pathophysiology, and addresses the risk factors and strategies for prevention.

Antioxidant or neurotrophic factor treatment preserves function in a mouse model of neovascularization-associated oxidative stress

In several disease states, abnormal growth of blood vessels is associated with local neuronal degeneration. This is particularly true in ocular diseases such as retinal angiomatous proliferation (RAP) and macular telangiectasia (MacTel), in which, despite the absence of large-scale leakage or hemorrhage, abnormal neovascularization (NV) is associated with local neuronal dysfunction. We describe here a retinal phenotype in mice with dysfunctional receptors for VLDL (Vldlr-/- mice) that closely resembles human retinal diseases in which abnormal intra- and subretinal NV is associated with photoreceptor cell death. Such cell death was evidenced by decreased cone and, to a lesser extent, rod opsin expression and abnormal electroretinograms. Cell death in the region of intraretinal vascular abnormalities was associated with an increased presence of markers associated with oxidative stress. Oral antioxidant supplementation protected against photoreceptor degeneration and preserved retinal function, despite the continued presence of abnormal intra- and subretinal vessels. What we believe to be novel, Müller cell-based, virally mediated delivery of neurotrophic compounds specifically to sites of NV was also neuroprotective. These observations demonstrate that neuronal loss secondary to NV can be prevented by the use of simple antioxidant dietary measures or cell-based delivery of neurotrophic factors, even when the underlying vascular phenotype is not altered.

RNA-Based Therapeutics: Ready for Delivery?

Delivery of RNA-based therapeutics to specific tissues for treating a variety of diseases faces many hurdles. But with several clinical trials and a slew of studies in animal models underway, the future of RNA-based therapeutics seems bright.

Diabetic Retinopathy and Angiogenesis

Diabetic retinopathy, a secondary microvascular complication of diabetes mellitus is the leading cause of blindness in the Unites States amongst individuals age 20 to 64. Two major retinal problems cause most of the diabetes-related vision loss: diabetic macular edema and complications from abnormal retinal blood vessel growth, angiogenesis. Secondary to angiogenesis, increased retinal blood flow is of pathogenic importance in the progression of diabetic retinopathy. Understanding the role of hyperglycemia seems to be the most critical factor in regulating retinal blood flow, as increased levels of blood glucose are thought to have a structural and physiological effect on retinal capillaries causing them to be both functionally and anatomically incompetent. High blood glucose induces hypoxia in retinal tissues, thus leading to the production of VEGF-A (vascular endothelial growth factor protein). Hypoxia is a key regulator of VEGF-induced ocular neovascularization. Secondary to the induction of VEGF by hypoxia, angiogenesis can be controlled by angiogenic inducers and inhibitors. The balance between VEGF and angiogenic inhibitors may determine the proliferation of angiogenesis in diabetic retinopathy. Since VEGF-A is a powerful angiogenic inducer, utilizing anti-VEGF treatments has proved to be a successful protocol in the treatment of proliferative diabetic retinopathy.

Corneal Lymphangiogenesis: Implications in Immunity

The lymphatic system is a network of vessels throughout the body that serves to return lymphatic fluid to the systemic blood circulation and serves as the immune system's highway in the afferent limb of the immune response. This paper reviews the basics of structure, function and development of the lymphatic system. The emphasis is on understanding the role of lymphatics in the cornea as related to angiogenesis and the clinical setting of corneal transplant rejection.The lymphatic system has two main functions: to drain excess extracellular fluid from capillary beds to return to the systemic blood circulation and to capture antigen for presentation to the immune system in the lymphoid compartment (e.g. nodes) connected in parallel to the lymphatic system. The normal cornea is devoid of lymphatic vessels and blood vessels, thus allowing for its unique immune privileged status. Abnormal blood vessel growth into the cornea has been studied for many years, but the study of lymphatic vessels has been hampered until the recent discovery of lymphatic-specific markers. Subsequent studies of vascularized corneas support the presence of lymphatic vessels in the cornea. The growth of new lymphatic (lymphangiogenesis) and blood (hemangiogenesis) vessels in the cornea is closely related to the abrogation of the immune privileged status of the cornea. This review summarizes our current understanding of the parallel development of hemangiogenesis and lymphangiogenesis as related to the mechanisms of immune rejection of corneal transplants.

Persistent Expression of PEDF in the Eye Using High-capacity Adenovectors

Ocular neovascularization, the growth of abnormal blood vessels in the eye, is a factor shared by the most common blinding diseases in developed countries. Pigment epithelium-derived factor (PEDF) is a potent antiangiogenic and neuroprotective protein that is normally produced in the eye. When delivered via an adenovector, PEDF can block the growth of new blood vessels and trigger the selective regression of abnormal vessels in animal models of ocular disease. Because of the absence of adenoviral genes, high-capacity (HC) adenovectors offer the potential for persistent transgene expression and enhanced tolerability. We have assessed the durability of PEDF expression and the induction of ocular inflammation following delivery of a PEDF-expressing HC adenovector compared to earlier generation vectors. The HC vector mediated prolonged PEDF expression in tissue-cultured pigmented epithelial cells and when delivered by intravitreal injection into the mouse eye. Delivery of first-generation adenovectors resulted in a dose-dependent increase in cytokine/chemokine gene expression, which correlated with the infiltration of inflammatory cells in the eye. In comparison, the levels of inflammatory gene expression and the intraocular infiltrate were substantially reduced following delivery of the HC vector. These results support the development of the HC adenovector gene delivery system for ocular disease.

Application Forum: Chemiluminescent VEGF ELISA Using the SpectraMax ® L Microplate Luminometer

BioTechniquesVol. 45, No. 5 Application Forum - Sponsored PaperOpen AccessChemiluminescent VEGF ELISA Using the SpectraMax® L Microplate LuminometerCathy OlsenCathy OlsenMDS Analytical Technologies, 1311 Orleans Drive, Sunnyvale, CA, 94089Search for more papers by this authorPublished Online:16 May 2018https://doi.org/10.2144/000113044AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail IntroductionVascular endothelial growth factors (VEGFs) are a family of secreted polypeptides implicated in mammalian vascular development and in disease processes involving abnormal blood vessel growth.(1) The QuantiGlo Chemiluminescent VEGF Immunoassay is a solid phase ELISA that uses the quantitative sandwich enzyme immunoassay technique to measure VEGF165 levels in cell culture supernatants, serum, plasma, saliva, and urine.(2) Luminescent signal is produced in proportion to the amount of VEGF present in samples and is measured using a microplate luminometer. Here, assay results were detected using the SpectraMax® L microplate luminometer, which performs flash and glow applications in 96- and 384-well microplate formats. Its dedicated luminescence optical design yields an extremely high signal-to-noise ratio, low crosstalk, and a dynamic range of over eight orders of magnitude. Data analysis was performed using a preconfigured protocol in the SoftMax® Pro software.MaterialsQuantiGlo Human VEGF Immunoassay (R&D Systems P/N QVEOOB).SpectraMax L microplate luminometer.MethodsPreparation of reagents and standardsStep 1. All reagents were brought to room temperature.Step 2. Wash Buffer Concentrate was diluted 10-fold with deionized water.Step 3. Working Glo Reagent was prepared as directed in the product insert.Step 4. The VEGF standard was reconstituted as directed. Working standards ranging from 20,000 to 1.3 pg/mL were prepared by serially diluting the stock standard in Calibrator Diluent RD5L.Assay procedureStep 1. 150 µL of Assay Diluent RD1-8 was pipetted into each well.Step 2. 50 µL of working standard or diluent was pipetted into triplicate wells of the supplied microplate. The microplate was incubated for 2 hours at room temperature with shaking.Step 3. Wells were washed 4 times with 400 µL Wash Buffer.Step 4. 200 µL of VEGF Conjugate was added to each well, and the microplate was incubated for 3 hours at RT with shaking.Step 5. The plate was washed as in step 3.Step 6. 100 µL Working Glo Reagent was added to each well. The microplate was covered and incubated for 5-20 minutes at RT.Step 7. The microplate was read on the SpectraMax L microplate luminometer using 1-second integration, AutoRange PMT, and 470 nm target calibration wavelength.ResultsFigure 1 shows the VEGF standard curve obtained with the SpectraMax L microplate luminometer. The dynamic range for this assay spanned greater than four orders of magnitude, and the calculated sensitivity of about 1.5 pg/mL correlated closely with R&D Systems’ stated value of 3.3 pg/mL. Data were analyzed and plotted using SoftMax Pro software. Similar results may be obtained using the SpectraMax M5 and FlexStation® 3 multi-mode microplate readers (data not shown).Figure 1. VEGF Standard Curve. Standards ranging from 20,000 to 1.3 pg/mL were assayed as described in the assay procedure. The microplate was read on a SpectraMax L microplate luminometer with a 1-second integration time and target calibration wavelength of 470 nm. Results were plotted using SoftMax Pro software.SummaryThe QuantiGlo Human VEGF Immunoassay is a highly sensitive method for assaying VEGF in a variety of samples. The SpectraMax L microplate luminometer with SoftMax Pro software provides excellent sensitivity and dynamic range for this assay as well as simplified data acquisition and analysis.

Repeat Administration of Proteins to the Eye With a Single Intraocular Injection of an Adenovirus Vector

Delivery of therapeutic proteins, such as antiangiogenic proteins, to the eye is a demonstrated method for the control of age-related macular degeneration (AMD). However, one of the key limitations is the requirement for frequent and repeated intraocular injections. In this article, we demonstrate that repeated protein production in the eye can be stimulated from the cytomegalovirus (CMV) promoter without repeat intraocular injections using a small molecule, all-trans retinoic acid (ATRA). ATRA by systemic delivery can stimulate protein production multiple times in the eye. Administration of ATRA resulted in stimulation of gene expression to relevant levels that block abnormal blood vessel growth in an experimental animal model for AMD. These data support the principles of this technological discovery to therapeutic applications for chronic ocular diseases.

Ranibizumab: the evidence of its therapeutic value in neovascular age-related macular degeneration

Neovascular age-related macular degeneration (AMD) is the leading cause of severe, irreversible visual impairment in people over 60 years of age. Neovascular AMD is characterized by abnormal growth of blood vessels under the retina, specifically the macula. These vessels leak blood and fluids, damaging the retina and its photoreceptors, resulting in permanent loss of central vision. Vascular endothelial growth factor-A (VEGF-A) has been shown to play a critical role in the pathogenesis of neovascular AMD. In the US, ranibizumab, a VEGF-A blocker, is approved and indicated for the treatment of patients with neovascular AMD. To review the clinical evidence for ranibizumab in the treatment of neovascular AMD. Phase III clinical trial data have established ranibizumab as a safe and well-tolerated treatment for neovascular AMD. Monthly intravitreal injections of ranibizumab result in a statistically significantly greater proportion of patients losing <15 letters of visual acuity (VA) and statistically significant increases in the mean number of letters gained compared with controls. Anatomically, ranibizumab results in stabilization in the mean area of choroidal neovascularization (CNV) and statistically significant reductions in the mean area of leakage compared with controls. Although there is limited economic evidence available, ranibizumab therapy for neovascular AMD appears to deliver a significant degree of value gain in terms of quality of life when compared with other neovascular AMD interventions. Clinical evidence establishes ranibizumab as a first-line therapy option for virtually all treatable neovascular AMD patients. Updating neovascular AMD treatment guidelines to reflect the evidence base for ranibizumab as a preferred first-line therapy would be beneficial for physicians in making informed treatment choices and ultimately helping to ensure the best care for patients.

Senescence regulates macrophage activation and angiogenic fate at sites of tissue injury in mice

Abnormal angiogenesis plays a key role in diseases of aging such as heart disease, certain cancers, and eye diseases including age-related macular degeneration. Macrophages have been shown previously to be both anti- and proangiogenic, and their role in regulating angiogenesis at sites of tissue injury is critical and complex. In this study, we analyzed cytokine gene expression patterns of mouse macrophages by real-time quantitative PCR and tested the functional effects of senescence on gene expression and macrophage polarization. Following laser injury to the retina, IL-10 was upregulated and Fas ligand (FasL), IL-12, and TNF-alpha were downregulated in ocular macrophages of old mice (>18 months of age). Downregulation of FasL on macrophages led to a loss of the antiangiogenic phenotype, as evidenced by the inability of these macrophages to inhibit vascular endothelial cells. Our results demonstrate that senescence, FasL, and IL-10 are key determinants of macrophage function that alter the growth of abnormal postdevelopmental blood vessels in disease processes including blinding eye disease.

Tumor refractoriness to anti-VEGF treatment is mediated by CD11b+Gr1+ myeloid cells

Vascular endothelial growth factor (VEGF) is an essential regulator of normal and abnormal blood vessel growth. A monoclonal antibody (mAb) that targets VEGF suppresses tumor growth in murine cancer models and human patients. We investigated cellular and molecular events that mediate refractoriness of tumors to anti-angiogenic therapy. Inherent anti-VEGF refractoriness is associated with infiltration of the tumor tissue by CD11b+Gr1+ myeloid cells. Recruitment of these myeloid cells is also sufficient to confer refractoriness. Combining anti-VEGF treatment with a mAb that targets myeloid cells inhibits growth of refractory tumors more effectively than anti-VEGF alone. Gene expression analysis in CD11b+Gr1+ cells isolated from the bone marrow of mice bearing refractory tumors reveals higher expression of a distinct set of genes known to be implicated in active mobilization and recruitment of myeloid cells. These findings indicate that, in our models, refractoriness to anti-VEGF treatment is determined by the ability of tumors to prime and recruit CD11b+Gr1+ cells.

Study Aims to Clarify Efficacy, Safety of Eye Drug Treatments

THE PRESS RELEASE SEEMS SIMPLE enough: “The National Eye Institute of the National Institutes of Health will fund a new multicenter clinical trial to compare 2 drugs currently used to treat advanced agerelated macular degeneration (AMD).” But its ramifications are enormous. Thedrugsinquestionareranibizumab (Lucentis) and bevacizumab (Avastin), bothmanufacturedbyGenentechInc,of SouthSanFrancisco.Currently,only ranibizumab is approved by the US Food andDrugAdministration(FDA)fortreatingneovascularAMD.Anditworkswell, with more than 90% of patients reportingahalt invision lossandabout30%reporting improved sight. But a single dose of ranibizumab costs almost $2000 (or $48 000 for a typical 2-year course of monthly treatments). Although Medicare pays for ranibizumab, it covers only 80% of the cost, meaning that patients who do not have coinsurance pay $9600 out of pocket for a 2-year course. (Genentech does provide financial assistance and a free drug program for those who qualify who cannot pay for treatment.) As an alternative, a significant number of physicians are using bevacizumab off-label for AMD treatment. It costs about $40 a dose. Payers such as Medicare and patients who lack insurance or have copays will obviously experience huge cost savings if bevacizumab is found to be as effective as ranibizumab. Such a finding would also provide another treatment option for AMD in developing countries, where ranibizumab is generally not affordable. On the flip side, Genentech stands to lose billions of anticipated dollars if ranibizumab is abandoned for AMD treatment. ButbeyondGenentech,thebiotechand drug manufacturing industries are concernedabouttheprecedentcreatedbythis trial andcoulduse theirpolitical clout in an attempt to derail the study, said William Rich III, MD, medical director of healthpolicy for theAmericanAcademy of Ophthalmology in Washington, DC. “Obviously, biotech hates head-tohead trials based on efficacy and affecting payment,” Rich said. “I’m anticipating delays for this trial.” Indeed, while the $16.2 million trial was announced last year, and published reports had the study beginning in May or June, now researchers involved only say the trial will begin “sometime this summer.” Inexplainingwhy it isnotconducting orparticipating in this trial, aGenentech spokeswomansaidthecompanybelieves it has invented a highly successful treatment inranibizumab,anditwouldrather devoteitsresourcestoexplorewaysofexpanding the drug’s use for other eyerelatedissuessuchasdiabeticretinopathy. Age-relatedmaculardegenerationisthe leading cause of blindness in the developed world among people aged 50 years or older. In the United States, the prevalenceofpeoplewithvisuallossfromAMD is 1.2 million with about 200 000 new casesbeingreportedannually.About7.3 millionpeople in theUnitedStates areat risk of developing the condition. The neovascular form of AMD is characterized by abnormal growth of new blood vessels involving the macula. Scientists believe that vascular endothelial growth factor (VEGF)promotes thisneovascularization. Ranibizumab and bevacizumab are monoclonal antibodies that neutralize VEGF. Researchers originally considered using bevacizumab as a treatment for AMD, but it was thought that the molecule created was too big to penetrate the retina. Bevacizumab went on to become, in 2004, the first angiogenesis inhibitor approved by the FDA for treatment of colorectal cancer. In the meantime, researchers created ranibizumab by reengineering bevacizumab, resulting in a smaller molecule capable of passing through the retina. Butbefore ranibizumabreceivedFDA approval last year,physicians, facingpatientsdesperatetoavoidblindness,turned to bevacizumab, which they found improvedvisionwhilebeingwell tolerated. Soonpharmacistsweresubdividingquantities of bevacizumab intended for colorectalcancertreatmentintosmallerdoses forAMD.Today,anecdotalevidencesuggestsabouthalfofallpatientsbeingtreated for AMD receive bevacizumab. With so many patients receiving a drug off-label with anecdotally reported success, it is important to determine whether bevacizumab truly is safe and effective for AMD treatment, said Daniel Martin, MD, who will lead the National Eye Institute study.

The ADMA/DDAH pathway is a critical regulator of endothelial cell motility

Asymmetric dimethylarginine (ADMA) is an inhibitor of nitric oxide production associated with abnormal blood vessel growth and repair, however, the mechanism of action of ADMA is not well understood. We studied the role of exogenous and endogenous ADMA in the regulation of cell motility and actin cytoskeleton in porcine pulmonary endothelial cells (PAECs) and pulmonary microvascular endothelial cells (PMECs) from knockout mice that lack one of the enzyme metabolising ADMA, dimethylarginine dimethylaminohydrolase I (DDAHI) as well as endothelial cells overexpressing DDAH in vitro. We show that ADMA induced stress fibre and focal adhesion formation and inhibited cell motility in primary pulmonary endothelial cells. The effects of ADMA depended on the activity of RhoA and Rho kinase and were reversed by overexpression of DDAH, nitric oxide donors and protein kinase G activator, 8-bromo-cGMP. ADMA also inhibited the activities of Rac1 and Cdc42 in cells but these changes had a minor effect on cell motility. Endogenous ADMA increased RhoA activity and inhibited cell motility in PMECs from DDAHI knockout mice and inhibited angiogenesis in vitro. These results are the first demonstration that metabolism of cardiovascular risk factor ADMA regulates endothelial cell motility, an important factor in angiogenesis and vascular repair.

Tumor-Induced Sentinel Lymph Node Lymphangiogenesis and Increased Lymph Flow Precede Melanoma Metastasis

Lymphangiogenesis is associated with human and murine cancer metastasis, suggesting that lymphatic vessels are important for tumor dissemination. Lymphatic vessel alterations were examined using B16-F10 melanoma cells implanted in syngeneic C57Bl/6 mice, which form tumors metastasizing to draining lymph nodes and subsequently to the lungs. Footpad tumors showed no lymphatic or blood vessel growth; however, the tumor-draining popliteal lymph node featured greatly increased lymphatic sinuses. Lymph node lymphangiogenesis began before melanoma cells reached draining lymph nodes, indicating that primary tumors induce these alterations at a distance. Lymph flow imaging revealed that nanoparticle transit was greatly increased through tumor-draining relative to nondraining lymph nodes. Lymph node lymphatic sinuses and lymph flow were increased in mice implanted with unmarked or with foreign antigen-expressing melanomas, indicating that these effects are not due to foreign antigen expression. However, tumor-derived immune signaling could promote lymph node alterations, as macrophages infiltrated footpad tumors, whereas lymphocytes accumulated in tumor-draining lymph nodes. B lymphocytes are required for lymphangiogenesis and increased lymph flow through tumor-draining lymph nodes, as these alterations were not observed in mice deficient for B cells. Lymph node lymphangiogenesis and increased lymph flow through tumor-draining lymph nodes may actively promote metastasis via the lymphatics.

Proliferating endothelial cell-specific expression of IGF-I receptor ribozyme inhibits retinal neovascularization

Insulin-like growth factor-I (IGF-I) and its receptor (IGF-IR) are essential for normal ocular development and are expressed in numerous ocular cell types including lens epithelial cells, retinal pigment epithelial cells, Müller cells and endothelial cells. Endothelial cell proliferation is a common feature of proliferative retinopathies and involves abnormal growth of blood vessels within and on the surface of the retina. In an effort to inhibit the formation of these aberrant blood vessels, we cloned an IGF-IR ribozyme into an expression vector that limits expression of the ribozyme to proliferating endothelial cells. An endothelin enhancer and Cdc6 promoter chimera drives expression of the IGF-IR ribozyme. This promoter limited retinal expression of the reporter gene to proliferating endothelial cells in two mouse models of proliferative retinopathy. In addition, expression of the IGF-IR ribozyme by this promoter inhibited aberrant retinal angiogenesis in both models while preserving normal vessels. These results demonstrate the feasibility of IGF-IR ribozyme expression in a selective manner for safer treatment of abnormal angiogenesis associated with retinopathy.