Co-culture Angiogenesis Assay Research Articles

In Vitro Coculture Assays of Angiogenesis.

During angiogenesis, endothelial cells must undergo a coordinated set of morphological changes in order to form a new vessel. There is a need for endothelial cells to communicate with each other in order to take up different identities in the sprout and to migrate collectively as a connected chord. Endothelial cells must also interact with a wide range of other cells that contribute to vessel formation. In ischemic disease, hypoxic cells in tissue will generate proangiogenic signals that promote and guide angiogenesis. In solid tumors, this function is co-opted by tumor cells, which make a complex range of interactions with endothelial cells, even integrating into the walls of vessels. In vessel repair, cells from the immune system contribute to the promotion and remodeling of new vessels. The coculture angiogenesis assay is a long-term in vitro protocol that uses fibroblasts to secrete and condition an artificial stromal matrix for tubules to grow through. We show here how the assay can be easily adapted to include additional cell types, facilitating the study of cellular interactions during neovascularization.

Dimethyl-2-oxoglutarate improves redox balance and mitochondrial function in muscle pericytes of individuals with diabetes mellitus

Aims/hypothesisTreatment of vascular complications of diabetes remains inadequate. We reported that muscle pericytes (MPs) from limb muscles of vascular patients with diabetes mellitus display elevated levels of oxidative stress causing a dysfunctional phenotype. Here, we investigated whether treatment with dimethyl-2-oxoglutarate (DM-2OG), a tricarboxylic acid cycle metabolite with antioxidant properties, can restore a healthy metabolic and functional phenotype.MethodsMPs were isolated from limb muscles of diabetes patients with vascular disease (D-MPs) and from non-diabetic control participants (ND-MPs). Metabolic status was assessed in untreated and DM-2OG-treated (1 mmol/l) cells using an extracellular flux analyser and anion-exchange chromatography–mass spectrometry (IC-MS/MS). Redox status was measured using commercial kits and IC-MS/MS, with antioxidant and metabolic enzyme expression assessed by quantitative RT-PCR and western blotting. Myogenic differentiation and proliferation and pericyte–endothelial interaction were assessed as functional readouts.ResultsD-MPs showed mitochondrial dysfunction, suppressed glycolytic activity and reduced reactive oxygen species-buffering capacity, but no suppression of antioxidant systems when compared with ND-MP controls. DM-2OG supplementation improved redox balance and mitochondrial function, without affecting glycolysis or antioxidant systems. Nonetheless, this was not enough for treated D-MPs to regain the level of proliferation and myogenic differentiation of ND-MPs. Interestingly, DM-2OG exerted a positive effect on pericyte–endothelial cell interaction in the co-culture angiogenesis assay, independent of the diabetic status.Conclusions/interpretationThese novel findings support the concept of using DM-2OG supplementation to improve pericyte redox balance and mitochondrial function, while concurrently allowing for enhanced pericyte–endothelial crosstalk. Such effects may help to prevent or slow down vasculopathy in skeletal muscles of people with diabetes.Graphical abstract

Adipose-derived mesenchymal stromal cells reverse high glucose–induced reduction of angiogenesis in human retinal microvascular endothelial cells

Background aimsDiabetic retinopathy (DR) is characterized by a progressive alteration of the retinal microvasculature, arising from microaneurysms to leaky vessels and finally abnormal neovascularization. The hyperglycemia-mediated loss of pericytes is a key event in vessel degeneration causing vascular destabilization. To overcome this, mesenchymal stromal cells (MSCs) have been tested as pericyte replacement in several animal models showing repair and regeneration of DR-damaged vasculature. MethodsWe hypothesized that adipose-derived mesenchymal stromal cells (ASCs) resist high glucose–induced challenges and protect human retinal microvascular endothelial cells (HRMVECs) from glucose-mediated injury. ASCs and HRMVECs were cultured under normal-glucose (NG; 1 g/L) and high-glucose (HG; 4.5 g/L) conditions comparing their phenotype and angiogenic potential. ResultsWhereas ASCs were generally unaffected by HG, HG caused a reduction of the angiogenic potential in HRMVEC. Indeed, HG-treated HRMVECs formed fewer vascular tube structures in a basement membrane angiogenesis assay. However, this was not observed in a direct ASC and HRMVEC coculture angiogenesis assay. Increased oxidative stress levels appeared to be linked to the HG-induced reduction of angiogenesis, which could be restored by ASC-conditioned medium and antioxidant treatment. ConclusionsThese findings suggest that ASC resist HG-stress whereas endothelial cell angiogenic capacity is reduced. Thus, ASC may be potentially therapeutically active in DR by restoring angiogenic deficits in retinal endothelial cells by the secretion of proangiogenic factors. However, these data also inquire for a thorough risk assessment about the timing of the ASC-based cell therapy, which can be considered advantageous at early stage of DR, but possibly detrimental at the late neo-angiogenic stage of DR.

Abstract 5757: Preclinical development and testing of a therapeutic antibody against LRG1

Abstract Introduction: The purpose of this study was to generate a therapeutic function-blocking antibody against leucine-rich alpha-2-glycoprotein 1 (LRG1), a novel regulator of pathological angiogenesis that is up-regulated in many cancers, and demonstrate efficacy in proof of principle experiments in mouse models. Experimental Procedures: Mice were immunized with recombinant human LRG1 and hybridomas generated. More than 100 monoclonal antibodies (mAbs) were first screened by surface plasmon resonance (SPR) to measure affinity for target, and then in a co-culture angiogenesis assay to evaluate functional activity. The most promising candidates were then tested in vivo in the mouse model of laser-induced choroidal neovascularization, and from this study a lead was humanized and deimmunized. The resulting antibody is a hinge-stabilized IgG4 that is now undergoing manufacturing and pre-clinical safety and toxicology testing. Results: Of the original set of mouse mAbs, an initial screen to eliminate low affinity (KD greater than 1nM) binders resulted in 25 progressing to functional testing in angiogenesis assays. The seven most effective blocking mAbs were then tested in vivo in the mouse model of laser-induced choroidal neovascularization. Intraocular injections of mAb yielded two that exhibited equivalent inhibition of neovascularization to a therapeutic VEGF-blocker, and of these, the higher affinity mAb (15C4) was selected for humanization. Epitope mapping identified an amino-acid sequence within human LRG1, that in the form of a synthetic peptide was able to compete the binding of 15C4 to LRG1. Multiple species sequence alignment revealed the epitope to be partially conserved with the corresponding mouse sequence, and SPR analysis showed that 15C4 binding to recombinant mouse LRG1 was several orders of magnitude lower than to human LRG1. Nevertheless, administration of 15C4 to tumor-bearing mice significantly inhibited tumor growth in a range of subcutaneous and genetic models. Furthermore, due to the vessel normalizing effects of 15C4, combinations of mAb with both immunotherapy and cytotoxics were significantly more effective at restricting tumor growth than either monotherapy. Conclusions: We have generated and characterized a novel vessel-normalizing, fully humanized function-blocking mAb against LRG1 that shows efficacy in mouse tumor models. GMP manufacture is on-going, and early stage clinical trials are planned in 2019. Financial Support: The work was supported by the Medical Research Council, the Rosetrees Trust and UCL Business. Citation Format: Stephen Moss, David Kallenberg, Vineeta Tripathi, Sterenn Davis, Jestin George, Marie O'Connor, Laura Dowsett, John Greenwood. Preclinical development and testing of a therapeutic antibody against LRG1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5757.

Abstract 3868: Development of a high-throughput screening co-culture angiogenesis assay system using hTERT immortalized primary cells

Abstract Angiogenesis is a multi-step physiological process which is involved in a large number of normal and disease state processes; In vitro angiogenesis models provide useful tools to study these processes, one of which is the analysis of tubule formation. Tubules formed in co-culture assays composed of both endothelial and stroma-producing cells were significantly more heterogeneous and more closely resembled capillaries than mono cell culture models utilizing only endothelial cells to generate tubules in an extracellular matrix. Current co-culture models using primary cells have donor variability and inconsistent results due to lot-to-lot variation. In this study, we established an in vitro co-culture model system consisting of an assay-ready mixture of an aortic endothelial cell line TeloHAEC-GFP and an hTERT immortalized adipose-derived mesenchymal stem cell line in a specially formulated medium containing VEGF supplement. Both cell lines were immortalized by hTERT alone and have been well-characterized; showing that the cells retain the most important characteristic of their primary counterparts. The new co-culture system forms functional tubular structures in less than 7 days, additionally, the hTERT-MSC cells which surround the tubular structures have undergone transformation indicated by elevated positive αSMA staining, indicating that the system has physiological relevance. Notably, our results showed the co-culture system has minimal lot-to-lot variation indicated by the treatment of three lots with the anti-cancer drug, ramucirumab. More importantly, the tubular formation efficiency is reduced or blocked by well-known anti-cancer drugs such as sunitinib and bevacizumab. We also tested four HIF-1 inhibitors identified in previous high-throughput screens and found that those compounds inhibited tubule formation in the co-culture system. These results suggest that the co-culture system can mimic the hypoxic environment in solid tumors. Previously the authors optimized the system for 384-well performance and here we report further optimization of the system into a 1536-well high-throughput format and a shortening of the assay time frame to 3 days. Using this format, we evaluated 2816 drugs from The National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection (NPC), and 35 potent inhibitors (IC50 ≤1 μM) were identified. Moreover, many known angiogenesis inhibitors were identified, such as topotecan, docetaxel, and bortezomib. Several potential novel angiogenesis inhibitors were also identified from this study. Among the inhibitors, some compounds were proven to be involved in the hypoxia-inducible factor-1α and the nuclear factor-kappa B pathways. These results demonstrate that the co-culture model described in this report provides a consistent and robust in vitro system for antiangiogenic drug screening. Citation Format: Chaozhong Zou, Shuaizhang LI, Chia-Wen Hsu, Menghang Xia, Metewo S. Enuameh. Development of a high-throughput screening co-culture angiogenesis assay system using hTERT immortalized primary cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3868.

Pericyte modulation by a functional antibody obtained by a novel single-cell selection strategy.

Pericytes surround the endothelial cells of the microvasculature where they serve as active participants in crucial vascular functions such as angiogenesis, stability, and permeability. However, pericyte loss or dysfunction has been described in a number of pathologies. Targeting pericytes could therefore prove instrumental in the further development of vascular therapeutics. To target the pericyte, a proteomic-based approach using antibody phage display was conducted. We present a novel single-cell selection strategy, with a modified selection step to drive the selection of antibodies toward relevant pericyte epitopes. Characterization of the selected antibodies revealed two antibodies with binding specificity for pericytes. The cognate antigen of one of the antibodies was identified as pericyte-expressed fibronectin. This antibody was shown to be a potent inhibitor of pericyte migration and to induce a pro-angiogenic response when included in a pericyte-endothelial cell co-culture angiogenesis assay. The selection method provides an efficient platform for the selection of functional antibodies which target pericytes. We obtain an antibody that interacts with a fibronectin epitope important for pericyte mobility and functionality. Targeting of this epitope in pathologies where pericytes are implicated could potentially be of therapeutic benefit.

Abstract 792: Development of high-throughput cell-based co-culture angiogenesis assay system using hTERT immortalized cells

Abstract Angiogenesis is a multi-step physiological process which is involved in a large number of normal and disease state processes; In vitro angiogenesis models provide very useful tools to study these processes, one of which is the analysis of tubule formation. Tubules formed in co-culture assays were significantly more heterogeneous and more closely resembled capillaries than Matrigel® tubules. Current co-culture models using primary cells have donor variability, and inconsistent results due to lot to lot variation. In this study, we established an in vitro co-culture model system consisting of an assay ready mixture of an aortic endothelial cell line TeloHAEC-GFP (hTERT immortalized human aortic endothelial cell line) and a hTERT immortalized adipose-derived mesenchymal stem cell line (hTERT-MSCs) in a specially formulated medium containing VEGF supplement (Angio-Ready™ Angiogenesis Assay System). Both cell lines were immortalized by hTERT (human telomerase reverse transcriptase) alone and have been well-characterized showing that the cells retain the most important characteristic of their parental counterparts. The new co-culture system forms functional tubular structures in less than 7 days, and in addition, the hTERT-MSC cells which surround the tubular structures have undergone transformation indicated by elevated positive αSMA staining (a marker of smooth muscle cells), indicating that the system has physiological relevance. Notably, our results showed the co-culture system has minimal lot-to-lot variation indicated by the treatment of three lots with the anti-cancer drug, Ramucirumab (Cyramza®), which also targets the VEGF pathway. Next, we tested the new system with compounds that impact angiogenesis, results demonstrated that the angiogenesis system responds positively to elevated doses of VEGF and negatively to increasing concentrations of suramin; more importantly, the tubular formation efficiency is reduced or blocked by well-known anti-cancer drugs such as Sunitinib (SUTENT®) and Bevacizumab (Avastin®), both of which target the VEGF pathway. Finally, we used the Angio-Ready™ system validated 4 HIF-1(hypoxia inducible factors-1) inhibitors which have anti-angiogenic properties identified by high-throughput screening methods; data showed the results of the new system match with other screening methods including a system screening time as short as 3 days. Therefore, the co-culture model developed by using hTERT-immortalized cell lines described in this report provide a consistent and robust in vitro system for studying cardiovascular biology, drug screening and tissue engineering. Citation Format: CHAOZHONG ZOU, Chia-Wen Hsu, Menghang Xia, Metewo S Enuameh. Development of high-throughput cell-based co-culture angiogenesis assay system using hTERT immortalized cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 792. doi:10.1158/1538-7445.AM2017-792

Abstract 356: Development and characterization of an in vitro co-culture angiogenesis assay system using hTERT immortalized cells for high throughput drug screening

Abstract Angiogenesis is a multi-step physiological process which is involved in a large number of normal and disease state processes; In vitro angiogenesis models provide very useful tools to study these processes, one of which is the analysis of tubule formation. Tubules formed in co-culture assays were significantly more heterogeneous and more closely resembled capillaries than Matrigel® tubules. Current co-culture models using primary cells have donor variability, and inconsistent results due to lot to lot variation. In this study, we established an in vitro co-culture model system consisting of an assay ready mixture of an aortic endothelial cell line TeloHAEC-GFP (hTERT immortalized human aortic endothelial cell line) and a hTERT immortalized adipose-derived mesenchymal stem cell line hTERT-MSC in a common specially formulated medium, the AngioReady™angiogenesis medium with VEGF supplement. Both cell lines were immortalized by hTERT (human telomerase reverse transcriptase) alone and have been well-characterized showing that the cells retain the most important characteristic of their parental counterparts. The new co-culture system forms functional tubular structures in less than 7 days, and in addition, the hTERT-MSC cells which surround the tubular structures have undergone transformation indicated by elevated positive αSMA staining (a marker of smooth muscle cells),indicating that the system has physiological relevance. Next, we tested the new system with compounds that impact angiogenesis, results demonstrated that the angiogenesis system responds positively to elevated doses of VEGF and negatively to increasing concentrations of suramin; more importantly, the tubular formation efficiency is reduced or blocked by well-known cancer drugs such as Sunitinib (SUTENT®) and Bevacizumab (Avastin®), both of which target the VEGF pathway. Notably, our results showed the co-culture system has minimal lot-to-lot variation indicated by the treatment of three lots of the Angio-ready™ system with the cancer drug, Ramucirumab (Cyramza®), which also targets the VEGF pathway. Finally, we used the Angio-ready™ system validated 4 HIF-1(hypoxia inducible factors-1) inhibitors which have anti-angiogenic properties identified by high-throughput screening methods; data showed the results of the new system match with other screening methods, what's more, Angio-ready™ system screening time can be as short as 3 days. Therefore, the co-culture models developed by using hTERT-immortalized cell lines described in this report provide a consistent and robust in vitro system for studying cardiovascular biology, drug screening and tissue engineering. Citation Format: Chaozhong Zou, Chia-Wen Hsu, Chengkang Zhang, Isabela Oliva, Metewo S. Enuameh. Development and characterization of an in vitro co-culture angiogenesis assay system using hTERT immortalized cells for high throughput drug screening. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 356.

In Vitro Coculture Assays of Angiogenesis.

During angiogenesis, endothelial cells invade into the stromal matrix: a complex, structured array of extracellular matrix proteins. This three-dimensional deformable substrate also contains a mixture of angiogenic factors as well as embedded stromal cells. Interactions between endothelial cells and the stromal tissue make complex and important contributions to the process of angiogenesis; however, the composition of the stromal matrix is hard to replicate in vitro. The coculture angiogenesis assay is a long-term assay that uses fibroblasts to secrete and condition a stromal matrix that more closely mimics tissue than a simple collagen gel. Like all in vitro assays of angiogenesis, it has both strengths and weaknesses. Here we give protocols for the two of the most useful applications of the assay: screening for regulators of angiogenesis and high-resolution imaging.

Beneficial Effects of Combined Antiangiogenesis and Heavy Ion Radiation Therapy

Beneficial Effects of Combined Antiangiogenesis and Heavy Ion Radiation Therapy

Suppression of Vascular Network Formation by Human Cardiac Endothelial Cells with Low Concentrations of Doxorubicin is Due to the Inhibition of Endothelial Cell Proliferation

Anticancer drug doxorubicin (DOX) is known to cause dilated cardiomyopathy in treated patients. We set out to examine if cardiac endothelial cells present a target of doxorubicin in the heart. Primary human cardiac microvascular endothelial cells (HCMVEC) form vascular network in vitro when co‐cultured with human cardiac fibroblasts (HCFB). DOX suppressed the formation of vascular networks in a co‐culture assay with an IC50 of 8 nM. DOX did not inhibit the production of VEGF by these cells at these low concentrations under both normoxic and hypoxic conditions, but caused moderate inhibition of VEGF release by hypoxic HCFB at concentrations above 250 nM. Similarly, it did not enhance apoptosis in these cells at the concentrations that were shown to completely suppress vascular sprouting in a co‐culture angiogenesis assay. We used Ki67 immunostaining to examine if DOX suppresses proliferation of HCMVEC in a co‐culture angiogenesis assay. Endothelial cell marker CD31 was used to identify HCMVEC. DOX markedly reduced numbers of CD31+ and CD31+Ki67+ cells in co‐cultures at concentrations above 4 nM. We conclude that HCMVEC are exceedingly sensitive to anti‐proliferative effect of DOX, and may present a viable target of this cardiotoxic drug in the heart. Endothelial effects of DOX are likely to contribute to the development of dilated cardiomyopathy. This work was supported by NIH NCRR grant 2P20RR016467‐09A1.

Fluorescent human co-culture angiogenesis assay: analysis of pro-angiogenic growth factors

BioTechniquesVol. 48, No. 3 Sponsored Paper - Application ForumOpen AccessFluorescent human co-culture angiogenesis assay: analysis of pro-angiogenic growth factorsDyke McEwen, Libby Oupicka, Eric Endsley & Vince GroppiDyke McEwen*Correspondence should be addressed to Dyke McEwen (E-mail Address: mcewen@essen-instruments.com)Essen Instruments, Ann Arbor Michigan, USASearch for more papers by this author, Libby OupickaEssen Instruments, Ann Arbor Michigan, USASearch for more papers by this author, Eric EndsleyEssen Instruments, Ann Arbor Michigan, USASearch for more papers by this author & Vince GroppiEssen Instruments, Ann Arbor Michigan, USASearch for more papers by this authorPublished Online:28 Jun 2018https://doi.org/10.2144/000113385AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail IntroductionAngiogenesis, which is the formation of new blood vessels from pre-existing vessels, is mandatory for tumor growth. Angiogenesis is also involved in other pathological disorders such as psoriasis and macular degeneration. The process can be divided into 3 stages: endothelial cell (EC) proliferation, EC migration and vessel morphogenesis. Essen's GFP-AngioKit, an in vitro co-culture assay system, recapitulates these three stages of the angiogenic cascade. The kinetic method described herein is contrasted to currently available methods in that it provides dynamic information not available from end point experiments.MethodsGFP-AngioKits were prepared by first expressing GFP in HUVECs and then co-culturing them in a 24-well microplate (Orange Scientific) with normal human dermal fibroblasts (NHDF) as previously described by Bishop et al. (1) and in collaboration with TCS CellWorks. The 24-well GFP-AngioKit was fed every 2–3 days and tubule formation was monitored by imaging the cultures every 12 hours in IncuCyte™ FLR. Essen's Angiogenesis Analysis Module was used to quantify tube length, tube area, and branch point formation over the time course of the experiment.ResultsIn the presence of VEGF, GFP-labeled HUVECs proliferate and migrate to form cell clusters by day 5 (Figure 1, A and B). Further treatment with VEGF and other pro-angiogenic factors, such as bFGF and EGF, promote tubule formation and lengthening from day 5 to 14 (Figure 1E). VEGF-stimulated cultures are fully mature by day 14 (Figure 1, C and D). Neutralizing antibodies to VEGF blocked the EGF-potentiation of VEGF, which establishes that EGF is acting via the VEGF pathway (Figure 1F). In the absence of VEGF, there is greatly diminished proliferation, migration, and tubule formation.Figure 1. Kinetic analysis of microvascular tubule formation.Panel A and C show IncuCyte FLR images of GFP-labeled tubes taken at day 5 and day 14, respectively. A corresponding computational analysis of tube formation using the Angiogenesis Analysis Module for day 5 and 14 is presented in panels B and D, respectively. The effect of VEGF, bFGF, and EGF on tube length over time is shown in panel E. Anti-VEGF antibody treatment inhibits VEGF-stimulated tube formation, presented in panel F. More information can be found at www.essen-instruments.com/angio.ConclusionCombining GFP-AngioKits with IncuCyte™ FLR enables kinetic and quantitative measurements of both the stimulation and inhibition of microvascular tubule formation. The GFP-AngioKit, in conjunction with IncuCyte™ FLR, can be used to screen for drugs affecting all stages of angiogenesis and to dissect signal transduction pathways.

Phosphatase inhibitors with anti-angiogenic effect in vitro

Sylvest L, Bendiksen CD, Houen G. Phosphatase inhibitors with anti-angiogenic effect in vitro. APMIS 2010; 118: 49–59.Levamisole has previously been identified as an inhibitor of angiogenesis in vitro and in vivo, but the mechanism behind the anti-angiogenic behavior has not yet been established. However, one known effect of levamisole is the inhibition of alkaline phosphatase, and this fact encouraged us to test other phosphatase inhibitors for their anti-angiogenic effects by using the same method as used to identify levamisole: an ELISA-based co-culture angiogenesis assay giving quantitative and qualitative results. Historically, intracellular phosphatases have been associated with the downregulation of signaling pathways, and kinases with their upregulation, but lately, the phospatases have also been coupled to positive signaling, which is why inhibition of phosphatases has become associated with anti-tumorigenic and anti-angiogenic effects. The results obtained in this work reveal several agents with anti-angiogenic potential and give a strong indication that phosphatase inhibition is linked to anti-angiogenic activity. An apparent disruption of endothelial tube formation was seen for seven of eight phosphatase inhibitors tested in the angiogenesis assay. By looking at the morphological results, it was seen that most of the inhibitors impaired proliferation and elongation of the endothelial cells, which still had a differentiated appearance. One inhibitor, PTP inhibitor IV, seemed to impair endothelial cell differentiation and induced the same morphology as when cells were treated with levamisole, although at a 200 times lower concentration than that of levamisole. Hence, our work points out compounds with a potential that may be of use in the search for new medical products for the treatment of malignant tumors, or other conditions where angiogenesis plays a central role.

Human primary co-culture angiogenesis assay reveals additive stimulation and different angiogenic properties of VEGF and HGF

Therapeutic angiogenesis aims to induce blood vessel growth in acute or chronic ischemic tissues and has gained tremendous interest over the last years. To study factors and combinations thereof that potentially induce or modify angiogenesis and to evaluate their therapeutic potential, various in vitro assays have been developed. Although endothelial cells have attracted most attention in these assays, they alone cannot complete vessel maturation since extracellular matrix (ECM) components and mesenchymal cells also play an important role in vascular development. To address this complexity we focussed on a human co-culture angiogenesis assay comprising primary endothelial cells as well as primary ECM-producing fibroblasts. In this assay HGF and VEGF as single factors and combined were tested for the potential to induce an angiogenic response, which was detected by image analysis assessing the area, length and branches of the formed vascular structures. The results show that the cytokines HGF and VEGF both promote angiogenesis in this co-culture assay by inducing distinguishable patterns of vascular structures. VEGF increases the length, area and branch point number of induced vessels whereas HGF mediates exclusively vascular area growth resulting in vascular structures of enlarged diameter. Moreover, the combination of both cytokines results in an additive increase of vascular diameter.

Human primary co-culture angiogenesis assay reveals additive stimulation and different angiogenic properties of VEGF and HGF

Therapeutic angiogenesis aims to induce blood vessel growth in acute or chronic ischemic tissues and has gained tremendous interest over the last years. To study factors and combinations thereof that potentially induce or modify angiogenesis and to evaluate their therapeutic potential, various in vitro assays have been developed. Although endothelial cells have attracted most attention in these assays, they alone cannot complete vessel maturation since extracellular matrix (ECM) components and mesenchymal cells also play an important role in vascular development. To address this complexity we focussed on a human co-culture angiogenesis assay comprising primary endothelial cells as well as primary ECM-producing fibroblasts. In this assay HGF and VEGF as single factors and combined were tested for the potential to induce an angiogenic response, which was detected by image analysis assessing the area, length and branches of the formed vascular structures. The results show that the cytokines HGF and VEGF both promote angiogenesis in this co-culture assay by inducing distinguishable patterns of vascular structures. VEGF increases the length, area and branch point number of induced vessels whereas HGF mediates exclusively vascular area growth resulting in vascular structures of enlarged diameter. Moreover, the combination of both cytokines results in an additive increase of vascular diameter.

A quantitative ELISA-based co-culture angiogenesis and cell proliferation assay.

Since solid tumours and metastases depend on adequate blood supply, much research is focused on inhibition of angiogenesis. Unfortunately, most known angiogenesis inhibitors have serious side effects when used as therapeutic agents in man. It is therefore important to develop methods to identify well-tolerated and efficient angiogenesis inhibitors. As a method for identification of new angiogenesis inhibitors we have further developed the procedure described by Bishop et al. (Angiogenesis 1999;3:335-44) to a quantitative ELISA-based fibroblast and endothelial cell co-culture angiogenesis assay. In each well of a 96-microwell plate, human umbilical vein endothelial cells (HUVEC) are seeded onto normal human dermal fibroblasts (NHDF) and propagated in co-culture for 72 h with or without a potential angiogenesis inhibitor. The effect on total cell proliferation is evaluated by quantitative immunochemical measurement of DNA, and on endothelial tube formation by quantification of CD 31, von Willebrand factor, and collagen IV. After ELISA reading, the morphology of the tubular structures formed by HUVEC is visualised with BCIP/NBT, permitting a quantitative result and a qualitative evaluation of cell morphology from the same well. We have used the assay to demonstrate the effect of well-known angiogenesis inhibitors on HUVEC tube formation.