Anti-vascular Drugs Research Articles

Level of endothelial cell apoptosis required for a significant decrease in microvessel density

Endothelial cell apoptosis plays a critical role in the disruption of blood vessels mediated by natural inhibitors of angiogenesis and by anti-vascular drugs. However, the proportion of endothelial cells required to mediate a significant decrease in microvessel density is unknown. A system based on an inducible caspase (iCaspase-9) offers a unique opportunity to address this question. The dimerizer drug AP20187 induces apoptosis of human dermal microvascular endothelial cells stably transduced with iCaspase-9 (HDMEC–iCaspase-9), but not control cells (HDMEC–LXSN). Here, we generated blood vessels containing several HDMEC–iCaspase-9:HDMEC–LXSN ratios, and developed a mathematical modeling involving a system of differential equations to evaluate experimentally inaccessible ratios. A significant decrease in capillary sprouts was observed when at least 17% of the endothelial cells underwent apoptosis in vitro. Exposure to vascular endothelial growth factor (VEGF 165) did not prevent apoptosis of HDMEC–iCaspase-9, but increased the apoptotic requirement for sprout disruption. In vivo experiments showed the requirement of at least 22% apoptotic endothelial cells for a significant decrease in microvascular density. The combined use of biological experimentation with mathematical modeling allowed us to conclude that apoptosis of a relatively small proportion of endothelial cells is sufficient to mediate a significant decrease in microvessel density.

A theoretical study of the response of vascular tumours to different types of chemotherapy

In this paper we formulate and explore a mathematical model to study continuous infusion of a vascular tumour with isolated and combined blood-borne chemotherapies. The mathematical model comprises a system of nonlinear partial differential equations that describe the evolution of the healthy (host) cells, the tumour cells and the tumour vasculature, coupled with distribution of a generic angiogenic stimulant (TAF) and blood-borne oxygen. A novel aspect of our model is the presence of blood-borne chemotherapeutic drugs which target different aspects of tumour growth (cf. proliferating cells, the angiogenic stimulant or the tumour vasculature). We run exhaustive numerical simulations in order to compare vascular tumour growth before and following therapy. Our results suggest that continuous exposure to anti-proliferative drug will result in the vascular tumour being cleared, becoming growth-arrested or growing at a reduced rate, the outcome depending on the drug’s potency and its rate of uptake. When the angiogenic stimulant or the tumour vasculature are targeted by the therapy, tumour elimination can not occur: at best vascular growth is retarded and the tumour reverts to an avascular form. Application of a combined treatment that destroys the vasculature and the TAF, yields results that resemble those achieved following successful treatment with anti-TAF or anti-vascular therapy. In contrast, combining anti-proliferative therapy with anti-TAF or antivascular therapy can eliminate the vascular tumour. In conclusion, our results suggest that tumour growth and the time of tumour clearance are highly sensitive to the specific combinations of anti-proliferative, anti-TAF and anti-vascular drugs.

Macromolecular MRI contrast agents for imaging tumor angiogenesis

Angiogenesis has long been accepted as a vital process in the growth and metastasis of tumors. As a result it is the target of several novel anti-cancer medications. Consequently, there is an urgent clinical need to develop accurate, non-invasive imaging techniques to improve the characterization of tumor angiogenesis and the monitoring of the response to anti-angiogenic therapy. Macromolecular MR contrast media (MMCM) offer this diagnostic potential by preferentially exploiting the inherent hyperpermeable nature of new tumor vessels compared with normal vessels. Over the last 10–15 years many classes of MMCM have been developed. When evaluated with dynamic contrast enhanced (DCE) MRI, a number of MMCM have demonstrated in vivo imaging properties that correlate with ex vivo histological features of angiogenesis. The enhancement patterns with some MMCM have been reported to correlate with tumor grade, as well as show response to anti-angiogenic and anti-vascular drugs. Future applications of MMCM include targeted angiogenesis imaging and drug delivery of anti-cancer ‘payloads’. Herein we discuss the best known MMCMs along with their advantages and disadvantages.

Targeting of the Antivascular Drug Combretastatin to Irradiated Tumors Results in Tumor Growth Delay

The aim of the study is to evaluate the effects of targeting the antivascular drug combretastatin to irradiated mouse melanomas. Combretastatin was incorporated into liposomes with surfaces modified by the addition of cyclo(Arg-Gly-Asp-D-Phe-Cys) (RGD) to create an immunoliposome (IL). This addition of RGD allows the liposome to be preferentially targeted to alphavbeta3, an integrin up-regulated in the vasculature of irradiated tumors. C57BL mice bearing a transplanted B16-F10 melanoma were randomly assigned to one of the following treatment groups: untreated, a single dose of 5-Gy radiation (IR), IL (14.5 mg/kg of combretastatin), 5-Gy radiation plus IL, and a systemic administration of free drug (81.0 mg/kg of combretastatin). In this transplanted tumor model, there was no significant increase in the volume of the IL + IR (5 Gy) treated tumors during the initial 6 days posttreatment; all other treatment groups exhibited exponential growth curves after day 3. The IL + IR (5 Gy) treatment resulted in a 5.1-day tumor growth delay compared to untreated controls. These findings indicate that preferential targeting of antivascular drugs to irradiated tumors results in significant tumor growth delay.

Phase I Trial of Combretastatin A-4 Phosphate with Carboplatin

Preclinical evidence of synergy led to a phase I trial employing combretastatin A-4 phosphate (CA4P), a novel tubulin-binding antivascular drug, in combination with carboplatin. Based on preclinical scheduling studies, patients were treated on day 1 of a 21-day cycle. Carboplatin was given as a 30-minute i.v. infusion and CA4P was given 60 minutes later as a 10-minute infusion. Sixteen patients with solid tumors received 40 cycles of therapy at CA4P doses of 27 and 36 mg/m(2) together with carboplatin at area under the concentration-time curve (AUC) values of 4 and 5 mg min/mL. The dose-limiting toxicity of thrombocytopenia halted the dose escalation phase of the study. Four patients were treated at an amended dose level of CA4P of 36 mg/m(2) and carboplatin AUC of 4 mg min/mL although grade 3 neutropenia and thrombocytopenia were still observed. Three lines of evidence are adduced to suggest that a pharmacokinetic interaction between the drugs results in greater thrombocytopenia than anticipated: the carboplatin exposure (as AUC) was greater than predicted; the platelet nadirs were lower than predicted; and the deviation of the carboplatin exposure from predicted was proportional to the AUC of CA4, the active metabolite of CA4P. Patient benefit included six patients with stable disease lasting at least four cycles. This study of CA4P and carboplatin given in combination showed dose-limiting thrombocytopenia. Pharmacokinetic/pharmacodynamic modeling permitted the inference that altered carboplatin pharmacokinetics caused the increment in platelet toxicity.

Antivascular cancer treatments: functional assessments by dynamic contrast-enhanced magnetic resonance imaging

New anticancer therapeutics that target tumor blood vessels promise improved efficacy and tolerability in humans. Early phase 1 drug trials have shown that the maximum tolerated dose may be inappropriate for more advanced clinical studies with efficacy endpoints. More advanced clinical trials have demonstrated that morphologic assessments of tumor response are of limited value for gauging the efficacy of treatment. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) can serve as pharmacodynamic indicator of biological activity for antivascular cancer drugs by helping to define the biologically active dose. DCE-MRI studies may also predict the efficacy of treatment on the basis of changes observed. If DCE-MRI is to be used for the selection of antivascular drugs that advance into efficacy trials, then it will be necessary to develop standardized approaches to measurement and robust analytic approaches with clear accepted endpoints specified prospectively that have biological validity. Such developments will be essential for multicenter trials in which it will be necessary to establish effective cross-site standardization of measurements and evaluation.

149 Inhibition of vasculogenic mimicry in melanoma by the antivascular drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA)

149 Inhibition of vasculogenic mimicry in melanoma by the antivascular drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA)

Dynamic magnetic resonance imaging of tumor perfusion

This work presents approaches and biomedical challenges of dynamic contrast enhanced magnetic resonance imaging (DCE-MRI). DCE-MRI using small molecular weight gadolinium chelates enables noninvasive imaging characterization of tissue vascularity. Depending on the technique used, data reflecting tissue perfusion (blood flow, blood volume, mean transit time), microvessel permeability surface area product, and extracellular leakage space can be obtained. Insights into these physiological processes can be obtained from inspection of kinetic enhancement curves or by the application of complex compartmental modeling techniques. Potential clinical applications include screening for malignant disease, lesion characterization, monitoring lesion response to treatment, and assessment of residual disease. Newer applications include prognostication, pharmacodynamic assessments of antivascular anticancer drugs, and predicting efficacy of treatment. For dynamic MRI to enter into widespread clinical practice, it will be necessary to develop standardized approaches to measurement and robust analysis approaches. These include the need for commercial equipment manufacturers to provide robust methods for rapidly measuring time-varying change in T1 relaxation rates, incorporation of arterial input function into kinetic modeling processes, robust analysis software that allows input from a variety of MRI devices, and validated statistical tools for the evaluation of heterogeneity.

138 Enhancement of the action of the antivascular drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) by co-administration of non-steroidal anti-inflammatory drugs

138 Enhancement of the action of the antivascular drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) by co-administration of non-steroidal anti-inflammatory drugs

Single Dose of the Antivascular Agent, ZD6126 (N-Acetylcoichinol-O-Phosphate), Reduces Perfusion for at Least 96 Hours in the GH3 Prolactinoma Rat Tumor Model

Tumor vasculature is an attractive therapeutic target as it differs structurally from normal vasculature, and the destruction of a single vessel can lead to the death of many tumor cells. The effects of antivascular drugs are frequently short term, with regrowth beginning less than 24 hours posttreatment. This study investigated the duration of the response to the vascular targeting agent, ZD6126, of the GH3 prolactinoma, in which efficacy and dose-response have previously been demonstrated. GH3 prolactinomas were grown in the flanks of eight Wistar Furth rats. All animals were treated with 50 mg/kg ZD6126. The tumors were examined with dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) 24 hours pretreatment and posttreatment, and at a single time between 48 and 96 hours posttreatment. No evidence of recovery of perfusion was observed even at the longest (96-hour) time point. Involvement of a statistician at the project planning stage and the use of DCE-MRI, which permits noninvasive quantitation of parameters related to blood flow in intact animals, allowed this highly significant result to be obtained using only eight rats.

Perfusion MR Imaging of Extracranial Tumor Angiogenesis

Dynamic contrast-enhanced MRI (DCE-MRI) using small molecular weight gadolinium chelates enables noninvasive imaging characterization of tissue vascularity. Depending on the technique used, data reflecting tissue perfusion (blood flow, blood volume, mean transit time), microvessel permeability surface area product, and extracellular leakage space can be obtained. Insights into these physiological processes can be obtained from inspection of kinetic enhancement curves or by the application of complex compartmental modeling techniques. Combining morphologic and kinetic features can increase the accuracy of clinical diagnoses. Potential clinical applications include screening for malignant disease, lesion characterization, monitoring lesion response to treatment, and assessment of residual disease. Newer applications include prognostication, pharmacodynamic assessments of antivascular anticancer drugs, and predicting efficacy of treatment. For dynamic MRI to enter into widespread clinical practice, it will be necessary to develop standardized approaches to measurement and robust analysis approaches.

Measuring tumour vascular response to antivascular and antiangiogenic drugs.

The tumour vasculature is an attractive target for therapy because of its accessibility to blood-borne anticancer agents and the reliance of most tumour cells on an intact vascular supply for their survival. For convenience, therapeutic targeting of the tumour vasculature can be divided into antiangiogenic approaches, which target the process of new blood vessel development and antivascular approaches, which target the established tumour vasculature. Many agents are now in clinical trial for the treatment of cancer by these methods. The main aim of this article is to describe the vascular effects of some of these agents and identify suitable end-points for measuring efficacy in early clinical trials. For drugs which are active below their maximum tolerated dose (MTD), measurement of vascular end-points is required to determine the most effective dosing/scheduling protocols. In addition, many of the current and developing antiangiogenic agents have additional mechanisms of action unrelated to angiogenesis per se, requiring measurement of vascular end-points to understand their mechanisms of action. Measurement of tumour microvascular density (MVD) from tumour biopsies is a common method for assessing the efficacy of antiangiogenic drugs. The limitations of this method and alternative end-points, which take into account vascular function, are discussed. Pre-clinical data regarding tumour response to the antivascular agent combretastatin A-4 3-0-phosphate (CA-4-P) are discussed in the context of guiding clinical trial planning. Finally, the accessibility of vascular end-points for clinical imaging is addressed.

MRI for assessing antivascular cancer treatments.

Selective antiangiogenesis and vascular targeting drugs hold out the promise of improved efficacy and tolerability for anticancer treatments. Early phase 1 drug trials have shown good tolerability for antiangiogenesis agents with biological activity below the maximum tolerated dose. Advanced clinical trials have demonstrated that morphological assessments of tumour response are of limited value in gauging the efficacy of treatment. MRI is a versatile technique which is sensitive to contrast mechanisms that can be affected by antivascular treatments; this use for MRI has been validated in xenografts and humans. Dynamic contrast-enhanced MRI (DCE-MRI), which demonstrates tissue perfusion and permeability, is being used clinically as a pharmacodynamic indicator of biological activity for antivascular cancer drugs. Early data show that DCE-MRI studies can define the biologically active dose and predict the efficacy of treatment on the basis of changes observed. MRI with macromolecular contrast media (MMCM) depicts microvessel permeability and fractional plasma volume. Xenograft studies with MMCM have shown great promise for evaluating antivascular treatments but this has not been used clinically. Intrinsic susceptibility-weighted MRI, which is sensitive to blood oxygenation and flow, is emerging as a technique that may be able to monitor vascular targeting therapies.

Molecular imaging using magnetic resonance: new tools for the development of tumour therapy

Molecular imaging - the exploitation of specific molecules as the source of image contrast - promises new insights into disease processes in the laboratory and since the imaging modalities employed are applicable clinically, can be used to translate this knowledge into new diagnostics and treatments in the clinic. This brief review focuses on the use of MR-based molecular imaging techniques for developing tumour therapy. As examples, methods for detecting drug-induced tumour cell apoptosis; the response of tumours and their susceptibilities to an antivascular drug; early signs of tumour immune rejection and methods for detecting immune cell infiltration of tumours are described.

Antivascular cancer treatments: imaging biomarkers in pharmaceutical drug development.

Over the last several years there have been a large number of drugs brought into the clinic for the treatment of cancer targeting antiangiogenic or antivascular mechanisms. The matrix metalloproteinase inhibitors (MMPIs) are an example of one group of antiangiogenic agents which were first tested in man beginning 1997 [1]. Pre-clinical studies had demonstrated inhibition of endothelial cell growth, inhibition of angiogenesis with matrigel plug assays, and inhibition of metastasis. There was no expectation from such studies though that these agents would cause tumour regression in the manner that cytotoxic drugs do. Thus, the pattern of development of oncology agents where in Phase I doses are escalated until dose limiting toxicity is seen and the next lowest dose is chosen as the maximum tolerated dose (MTD) was perceived to be inappropriate [2]. It was proposed that for drugs with a wider therapeutic window than traditional cytotoxics, and a mechanism of action that may lead to cytostasis, rather than tumour regression, that an ‘‘optimal biological dose’’ (OBD) lower than the MTD might be more appropriate to take forward into Phase II. However in order to discriminate the OBD some means of measuring the desired biological activity or ‘‘biomarker’’ is needed. At the time when the MMPIs entered the clinic very few such biomarkers were available. However some exploratory markers were examined by more recent entrants to the antiangiogenic field such as endostatin [3, 4], the vascular endothelial growth factor (VEGF) pathway targeted drugs [5‐8] and vascular targeting agents [11, 12]. In addition to biomarkers in tumour tissue and blood samples, a range of imaging technologies are available or under development that have the potential for use as biomarkers of antiangiogenic and antivascular drug effects. These can be categorized by the distance ‘‘downstream’’ from the drug interaction with its molecular target:

Differential relationship between changes in tumour size and microcirculatory functions induced by therapy with an antivascular drug and with cytotoxic drugs: implications for the evaluation of therapeutic efficacy of AC7700 (AVE8062)

A novel combretastatin A-4 derivative, AC7700, which is now in Phase I clinical trials under a new code, AVE8062, has shown strong antitumour effects against solid tumours in rodents because of its powerful and continued stanching of the tumour blood flow (TBF). Despite the strong tumour-suppressing qualities of AC7700, it does not produce an immediate reduction in tumour size. To elucidate the reason for this effect, we investigated the relationship between the change in tumour size in Sato lung carcinoma (SLC) and circulatory functions after therapy with AC7700, doxorubicin (Adriamycin [ADR]), or mitomycin C (MMC). To measure time-lapse changes in TBF with the hydrogen clearance method at the same site after drug administration, we developed a new apparatus for keeping electrodes within a tumour. AC7700 led to the destruction of both cancer cells and tumour vessels by interrupting the supply of nutrients. Intravenous (i.v.) administration of fluorescent dyes after AC7700 treatment revealed no fluorescence within the tumour vessels, which confirmed that the tumour microcirculation had been completely blocked. In contrast, ADR led to the destruction of SLC tumour cells, but did not have the same effect on tumour vessels. Intravenously administered fluorescent dyes immediately reached the tumour, which indicated that the tumour vasculature remained intact, and the TBF remained at the preadministration level, even 6 days after ADR treatment. In addition, although the size of the tumour increased slightly for 2 days with ADR treatment, possibly because of swelling of the cancer cells, thereafter it continued to decrease. MMC had virtually no effect on SLC tumour cells, tumour size or tumour vessels. We conclude that changes in tumour size brought about by cancer chemotherapy depend not only on the sensitivity of the cancer cells to the drug in question, but also on the nature of changes in the microcirculatory functions of the tumour brought about by the therapy. When both tumour cells and the tumour vasculature are destroyed, the effectiveness of therapy can not be determined from changes in tumour size alone.

Requirements for ICAM-1 immunogene therapy of lymphoma.

Intercellular cell adhesion molecule-1 (ICAM-1) is a cell-surface glycoprotein capable of eliciting bidirectional signals that activate signalling pathways in leukocytes, endothelial, and smooth muscle cells. Gene transfer of xenogeneic ICAM-1 into EL-4 lymphomas causes complete tumor rejection; however, it is unknown whether the mechanism responsible involves the "foreignness" of the ICAM-1 transgene, bidirectional signalling events, ICAM-1-receptor interaction, or a combination of the latter. To begin to address this question, we constructed four different therapeutic expression vectors encoding full-length ICAM-1, and forms in which the N-terminal ligand-binding domains and cytoplasmic tail had been deleted. Mouse EL-4 tumors (0.5 cm in diameter), which actively suppress the immune response, were significantly inhibited in their growth following injection of expression plasmids encoding either full-length xenogenic (human) ICAM-1, or a functional cytoplasmic domain-deficient form that retains ligand-binding activity. Efficacy of ICAM-1-mediated antitumor immunity was significantly augmented by administration of the antivascular drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA), which suppressed blood supply to the tumor, leading to enhanced leukocyte infiltration, and complete tumor eradication in a gene dosage and CD8(+) T cell and NK cell-dependent fashion. Generation of potent cytotoxic T cell (CTL)-mediated antitumor immunity was reflected by ICAM-1-facilitated apoptosis of tumor cells in situ. In contrast, nonfunctional ICAM-1 lacking the N-terminal ligand-binding Ig domain failed to generate antitumor immunity, even in the presence of DMXAA. These studies demonstrate that ICAM-1-stimulated antitumor immunity can overcome tumor-mediated immunosuppression, particularly when employed in combination with an attack on the tumor vasculature. The ligand-binding domain of ICAM-1 is essential for generating antitumor immunity, whereas the cytoplasmic domain and bidirectional activation of tumor signalling pathways are not essential.

Effect of 3-fluorothalidomide and 3-methylthalidomide enantiomers on tumor necrosis factor production and antitumor responses to the antivascular agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA).

5,6-Dimethylxanthenone-4-acetic acid (DMXAA) is an antivascular drug that induces tumor necrosis factor (TNF) in mice. Thalidomide inhibits TNF induction by DMXAA and also potentiates its antitumor activity. We investigated whether these effects were enantiomer specific, using the R- or S-enantiomers of two nonracemizable thalidomide analogues. Racemic 3-fluorothalidomide (3FThal) and racemic 3-methylthalidomide (3MeThal) were separated into enantiomers of greater than 98% optical purity using preparative chiral column chromatography. C57Bl/6 mice implanted with subcutaneous Colon 38 tumors were treated with DMXAA (25 mg/kg) alone or together with the pure R- or S-enantiomers by a single i.p. injection. TNF levels in the serum or tumor tissues 3 h after treatment were measured using ELISAs and tumor growth was also measured. 3FThal and 3MeThal, at their respective single maximum tolerated doses (MTD) of 15 and 50 mg/kg, were more toxic in mice than thalidomide (100 mg/kg). The R- and S-enantiomers of either 3FThal or 3MeThal, at their respective MTD, inhibited DMXAA-induced TNF activity in serum and tumor tissue, but no significant differences were observed between the enantiomers. Coadministration of racemic or enantiomers of 3FThal or 3MeThal at their respective MTD did not potentiate the antitumor responses above that obtained with DMXAA alone, and no enantioselectivity was apparent. We conclude that there is no advantage in using the nonracemizable thalidomide analogues to improve the antitumor activity of DMXAA.

The antitumour activity of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) in TNF receptor-1 knockout mice.

5,6-dimethylxanthenone-4-acetic acid, a novel antivascular anticancer drug, has completed Phase I clinical trial. Its actions in mice include tumour necrosis factor induction, serotonin release, tumour blood flow inhibition, and the induction of tumour haemorrhagic necrosis and regression. We have used mice with a targeted disruption of the tumour necrosis factor receptor-1 gene as recipients for the colon 38 carcinoma to determine the role of tumour necrosis factor signalling in the action of 5,6-dimethylxanthenone-4-acetic acid. The pharmacokinetics of 5,6-dimethylxanthenone-4-acetic acid, as well as the degree of induced plasma and tissue tumour necrosis factor, were similar in tumour necrosis factor receptor-1−/− and wild-type mice. However, the maximum tolerated dose of 5,6-dimethylxanthenone-4-acetic acid was considerably higher in tumour necrosis factor receptor-1−/− mice (>100 mg kg−1) than in wild-type mice (27.5 mg kg−1). The antitumour activity of 5,6-dimethylxanthenone-4-acetic acid (25 mg kg−1) was strongly attenuated in tumour necrosis factor receptor-1−/− mice. However, the reduced toxicity in tumour necrosis factor receptor-1−/− mice allowed the demonstration that at a higher dose (50 mg kg−1), 5,6-dimethylxanthenone-4-acetic acid was curative and comparable in effect to that of a lower dose (25 mg kg−1) in wild-type mice. The 5,6-dimethylxanthenone-4-acetic acid -induced rise in plasma 5-hydroxyindoleacetic acid, used to reflect serotonin production in a vascular response, was larger in colon 38 tumour bearing than in non-tumour bearing tumour necrosis factor receptor-1−/− mice, but in each case the response was smaller than the corresponding response in wild-type mice. The results suggest an important role for tumour necrosis factor in mediating both the host toxicity and antitumour activity of 5,6-dimethylxanthenone-4-acetic acid, but also suggest that tumour necrosis factor can be replaced by other vasoactive factors in its antitumour action, an observation of relevance to current clinical studies.British Journal of Cancer (2002) 87, 465–470. doi:10.1038/sj.bjc.6600479 www.bjcancer.com© 2002 Cancer Research UK

Tubes, Branches, and Pillars

The angiogenic cascade is getting increasingly complex. A few years ago, vasculogenesis and angiogenesis were considered as the primary mechanisms leading to the formation of new blood vessels. The original definition of vasculogenesis denotes the formation of a primary embryonic vascular network from in situ differentiating angioblastic cells.1 In contrast, angiogenesis primarily referred to the sprouting of blood vessels from preexisting vessels.1 Recent advances in the identification of molecules that regulate angiogenesis and vascular remodeling have shown that the simplistic model of an invading capillary sprout is not sufficient to appreciate the whole spectrum of morphogenic events that are required to form a neovascular network (Figure 1).1–3 Undoubtedly, vascular endothelial growth factor (VEGF) acts at an early point in the hierarchical order of morphogenic events and probably fulfills all criteria to be considered as a master switch of the angiogenic cascade. In contrast, the angiopoietins and their receptor Tie-2 as well as the ephrins and their corresponding Eph receptors appear to act at a somewhat later stage of neovessel formation. These molecules orchestrate a number of related, yet functionally and molecularly not well understood, processes such as vessel assembly (network formation and formation of anastomoses), vessel maturation (recruitment of mural cells [pericytes and smooth muscle cells], and extracellular matrix assembly, pruning of the primary vascular bed), and acquisition of vessel identity (formation of arteries, capillaries, and veins)3,4 (Figure 2). Lastly, the mechanisms of organotypic differentiation of the vascular tree (continuous endothelium, discontinuous endothelium, fenestrated endothelium) are not at all understood and the first molecules that govern subpopulation-specific vascular growth and differentiation are just being uncovered.5,6 Figure 1. Change of paradigm. From sprouting angiogenesis to vascular morphogenesis. Basement membrane degradation, directed endothelial cell migration, and proliferation (left) were considered as the primary mechanisms of angiogenesis. …