Results from this study indicate that a co-culture system of progenitor cells and endothelial cells can create a cord network with components found in the vasculature: endothelial cells, pericytes, and basement membrane. Exogenously added VEGF can stimulate cord formation and the cords that develop become insensitive to VEGF inhibition as the cords mature. An in vivo co-implant model of vasculogenesis, using the same progenitor and endothelial cells as the in vitro approach, develops functional blood vessels that anastamose with the host vasculature. These vessels also become insensitive to VEGF pathway inhibition with time. Together, these studies indicate that the combined use of an in vitro high-throughput established cord formation assay and an established in vivo co-implant model of vasculogenesis can be used to identify novel drugs that can target VEGF-independent blood vessels.
Here, we investigate two different in vitro models of angiogenesis. The first model uses co-cultures of human umbilical vein endothelial cells (HUVEC) with normal human dermal fibroblasts (NHDF) and the other uses co-cultures of endothelial colony forming cells (ECFC) with adipose-derived stem cells (ADSC). When seeded with NHDF, the HUVECs recapitulate the major phases of the angiogenic process, initially proliferating and migrating into endothelial clusters followed by differentiation and branching into complex networks over the 7–10 day assay. Under basal conditions, little tube formation occurs, while VEGF addition on day 3–4 stimulates tube formation in a concentration-dependent manner [37–39]. Further characterization of the HUVEC/NHDF approach shows that the pharmacological and physiological effects on endothelial cell biology are highly translatable to previous in vivo characterizations, exemplified by DLL4/Notch inhibition resulting in increased branch point formation late in the angiogenic process [39–41]. One of the major advantages of the ADSC and ECFC co-culture model is that the process of developing cords occurs quickly and incorporates a pericyte-like biology associated with the cords. Unlike the NHDF/HUVEC model and other similar models, in the ADSC/ECFC model, the majority of VEGF-driven cords are formed within the first 24 hours [23, 24, 38, 42]. Additional stimulation allowed for further remodeling of the vessels and differentiation of the ADSCs into SMA or PDGFR-β expressing pericyte-like cells. Characterization of these SMA associated cords indicates that drugs targeting VEGF or PDGF can inhibit the cords or the pericytes, respectively, similar to what has previously been shown in vivo[8, 28]. Further, VEGF is not the only growth factor that induces rapid cord formation. Other pro-angiogenic growth factors, including FGF and EGF, also induce cord formation in a concentration-dependent manner and exhibit different phenotypes and kinetics (manuscript under preparation). In addition to the assay duration and the cord similarities to in vivo vascular structure, the use of basal media in the ADSC/ECFC co-culture assay has dramatically increased the response window for drug screening purposes.
Interestingly, we found that inhibition of the VEGF receptor with sunitinib or ramucirumab leads to decreases in basal and VEGF driven cord formation, but inhibition of the ligand with bevacizumab only affected the VEGF driven cords. There may be several explanations for this effect. Endothelial cells can make VEGF and signal in an autocrine fashion. In fact, previous studies using endothelial cell specific knockout of VEGF indicates that autocrine VEGF signaling is required for the homeostasis of blood vessels . It is also feasible that ligand-independent mechanisms or signaling through heterodimerization of VEGFR2 with other receptors may play an important role in basal cord formation . Internalization of the receptor with ramucirumab or the multi-targeted nature of Sunitinib may play a role as well. Finally, VEGF secreted in the co-culture system may get bound to the extracellular matrix, where it may not be accessible to VEGF antibodies, but may still be able to be affected by receptor inhibition. These possibilities require further exploration as it is unclear what mechanism or mechanisms are involved in the ECFC/ADSC co-culture assay.
Unlike most tube and cord formation assays, established cords in the ADSC and ECFC co-culture system lose their dependence on VEGF once the cords are developed. Even after 1 day of establishment, the cords are less sensitive to multiple inhibitors of the VEGF signaling pathway. The mechanism of this VEGF-independence is not clear. Pericyte coverage is thought to make vessels insensitive to VEGF inhibition, but in this assay, we see increased VEGF independence after 1 day even though the SMA differentiation does not occur until day 3. In addition, inhibition of the PDGFR after 4 days of establishment decreased the SMA index but did not significantly alter total tube area. While it is still possible that pericytes play an important role in maintenance of established vessels, in this assay system other growth factors secreted by the ADSC feeder layer likely play a major role in maintaining the cords once they have been created. Clearly, there are some cords that can form without the addition of VEGF. Previous studies and our data indicate that HGF is highly expressed by the ADSCs and contributes to basal cord formation . In vivo studies show that inhibition of VEGF and the HGF receptor, c-Met, decrease tumor vessels more than VEGF inhibition alone . Together, these results indicate that the HGF secreted by the feeder layer may have an important role in maintenance of the established cords. In addition, we show that suramin, a broad-spectrum antagonist that inhibits various angiogenesis-related growth factors such as insulin-like growth factor, epidermal growth factor, platelet-derived growth factor, VEGF, and basic fibroblast growth factor, is able to reduce the VEGF-independent cords. Together, these results indicate that other growth factors secreted from the ECFCs or ADSC feeder layer may maintain cords following the initial VEGF stimulation.
Using an in vivo co-implant model of vasculogenesis with ADSCs and ECFCs, functional vessels can form after anastamosing with the host vasculature. These vessels form over 3 days and blood cells labeled with TER119 can be seen beginning 4 days after the cells are injected into the flank. Treatment of the vasculogenic plugs with a VEGF inhibitor dramatically decreases blood vessel formation if given at the beginning of the assay. If, however, VEGFR signaling was not inhibited until the vessels have established and have blood flow (at day 4), there is little effect. While the mechanism of this insensitivity is not know, it would be interesting to characterize our VEGF independent vessels to determine whether they have similar phenotypes as those described by the Dvorak laboratory [20, 21]. Nonetheless, these results are consistent with our high-throughput in vitro assay and provide a unique in vivo model to examine the effects of novel drugs on vessels that are insensitive to VEGF inhibition.
As proof of principle, a broad-spectrum anti-angiogenic inhibitor (suramin), a vascular disrupting agent (combretastatin), and a combination of a gamma secretase and VEGF inhibitor were tested on VEGF established cords. Suramin blocks a variety of growth factors including many angiogenesis-related factors. One of the proposed mechanisms of VEGF-independent tumor vessels is that inhibition of VEGF leads to induction of other proangiogenic factors [4, 14, 15, 34]. Suramin is likely able to block many of these other proangiogenic factors to reduce the cords in our established cord assay system.
The vascular disrupting agent combretastatin is a microtubule-depolymerizing agent which binds to tubulin dimers to prevent microtubule polymerization. This results in mitotic arrest and apoptosis of endothelial cells. In addition, combretastatin disrupts the endothelial cell junction molecule (VE-cadherin) leading to vascular collapse  and in vivo studies show that combretastatin is able to reduce immature vessels [45, 46]. We observed reductions in established cords with combretastatin treatment. Clearly, while combretastatin may not reduce all mature vessels in vivo, it is able to target a unique population of vessels or cords that are insensitive to VEGF inhibition. In fact, preclinical and clinical studies indicate that combining combretastatin with bevacizumab is more efficacious than either inhibitor alone [47, 48].
A recent in vivo study indicates that VEGF-independent vessels are driven by DLL4-Notch signaling and are sensitive to gamma secretase inhibition . Consistent with this novel strategy to overcome anti-angiogenic resistance, a gamma secretase inhibitor was tested in our in vitro and in vivo models alone or in combination with inhibition of VEGF signaling. In the in vitro system, treatment with either compound alone prevented a slight increase in cords associated with feeding the cells with fresh VEGF, but did not disrupt established networks. However, when VEGF and gamma secretase inhibitors were combined, there was a reduction in the number of cords. Similarly, in the in vivo co-implant model, ramucirumab or the gamma secretase inhibitor alone elicited a slight reduction in the vessels, but the combination reduced the vessels significantly more. These results indicate that our established cord assays may be used to identify new pathways involved in anti-VEGF/VEGFR directed therapy resistance and potential combinatorial strategies.
Many current angiogenesis assays used to screen anti-angiogenic agents are highly VEGF dependent. However, from preclinical and clinical analysis, there clearly exists a population of tumor vessels that are insensitive to VEGF inhibition. Thus, angiogenic assays are needed in which novel agents can be tested for their effectiveness on vessels which are not dependent on VEGF. The ECFC/ADSC assay is high throughput and relatively quick. Results can be obtained in approximately a week and can be run in 96-well and 384-well formats and similar co-culture approaches have previously been used in high-throughput drug discovery [24, 49]. In addition, labeling the ECFCs with GFP is a feasible approach to monitor cord formation and effects on established cords using continuous live-cell monitoring. Together, these results indicate that a co-culture cord formation system with ADSCs and ECFCs is a useful method to identify and characterize novel drugs on VEGF-independent cords. It would be interesting to identify selective markers on tumor vessels that remain after VEGF therapy and determine if the same markers exist in this co-culture system. If so, these in vitro and in vivo systems would be conducive to interrogate the mechanisms by which vessels become insensitive to VEGF inhibition though use of shRNA/siRNA knockdowns. With more and more studies being published regarding mechanisms of VEGF resistance, additional targets should be tested in this in vitro co-culture system.