First-in-Human Phase I Study of MP0250, a First-in-Class DARPin Drug Candidate Targeting VEGF and HGF, in Patients With Advanced Solid Tumors.

PURPOSE: A first-in-human study was performed with MP0250, a DARPin drug candidate. MP0250 specifically inhibits both vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) with the aim of disrupting the tumor microenvironment. PATIENTS AND METHODS: A multicenter, open-label, repeated-dose, phase I study was conducted to assess the safety, tolerability, and pharmacokinetics of MP0250 in 45 patients with advanced solid tumors. In the dose-escalation part, 24 patients received MP0250 as a 3-hour infusion once every 2 weeks at five different dose levels (0.5-12 mg/kg). Once the maximum tolerated dose (MTD) was established, 21 patients were treated with a 1-hour infusion (n = 13, 8 mg/kg, once every 2 weeks and n = 8, 12 mg/kg, once every 3 weeks) of MP0250 in the dose confirmation cohorts. RESULTS: In the dose-escalation cohort, patients treated with 12 mg/kg MP0250 once every 2 weeks experienced dose-limiting toxicities. Therefore, MTD was 8 mg/kg once every 2 weeks or 12 mg/kg once every 3 weeks. The most common adverse events (AEs) were hypertension (69%), proteinuria (51%), and diarrhea and nausea (both 36%); hypoalbuminemia was reported in 24% of patients. Most AEs were consistent with inhibition of the VEGF and HGF pathways. Exposure was dose-proportional and sustained throughout the dosing period for all patients (up to 15 months). The half-life was about 2 weeks. Signs of single-agent antitumor activity were observed: 1 unconfirmed partial response with a time to progression of 23 weeks and 24 patients with stable disease, with the longest duration of 72 weeks and a median duration of 18 weeks. CONCLUSION: MP0250 is a first-in-class DARPin drug candidate with suitable tolerability and appropriate pharmacokinetic properties for further development in combination with other anticancer therapies.

Profound Functional Suppression of Tumor-Infiltrating T-Cells in Ovarian Cancer Patients Can Be Reversed Using PD-1-Blocking Antibodies or DARPin® Proteins.

PD-1/PD-L1 blockade has revolutionized the field of immunooncology. Despite the relative success, the response rate to anti-PD-1 therapy requires further improvements. Our aim was to explore the enhancement of T-cell function by using novel PD-1-blocking proteins and compare with clinically approved monoclonal antibodies (mAbs). We isolated T-cells from the ascites and tumor of 17 patients with advanced epithelial ovarian cancer (EOC) and analyzed the effects using the mAbs nivolumab and pembrolizumab and two novel engineered ankyrin repeat proteins (DARPin® proteins). PD-1 blockade with either mAb or DARPin® molecule significantly increased the release of IFN-γ, granzyme B, IL-2, and TNF-α, demonstrating successful reinvigoration. The monovalent DARPin® protein was less effective compared to its bivalent equivalent, demonstrating that bivalency brings an additional benefit to PD-1 blockade. Overall, we found a higher fold increase of lymphokine secretion in response to the PD-1 blockade by tumor-derived T-cells; however, the absolute amounts were significantly lower compared to the release from ascites-derived T-cells. Our results demonstrate that PD-1 blockade can only partially reinvigorate functionally suppressed T-cells from EOC patients. This warrants further investigation preferably in combination with other therapeutics. The study provides an early pilot proof-of-concept for the potential use of DARPin® proteins as eligible alternative scaffold proteins to block PD-1.

Targeting angiogenesis in multiple myeloma by the VEGF and HGF blocking DARPin® protein MP0250: a preclinical study.

The investigational drug MP0250 is a multi-specific DARPin® molecule that simultaneously binds and neutralizes VEGF and HGF with high specificity and affinity. Here we studied the antiangiogenic effects of the MP0250 in multiple myeloma (MM). In endothelial cells (EC) isolated from bone marrow (BM) of MM patients (MMEC) MP0250 reduces VEGFR2 and cMet phosphorylation and affects their downstream signaling cascades. MP0250 influences the secretory profile of MMEC and inhibits their in vitro angiogenic activities (spontaneous and chemotactic migration, adhesion, spreading and capillarogenesis). Compared to anti-VEGF or anti-HGF neutralizing mAbs, MP0250 strongly reduces capillary network formation and vessel-sprouting in a Matrigel angiogenesis assay. MP0250 potentiates the effect of bortezomib in the same in vitro setting. It significantly reduces the number of newly formed vessels in the choriollantoic membrane assay (CAM) and the Matrigel plug assay. In the syngeneic 5T33MM tumor model, MP0250 decreases the microvessel density (MVD) and the combination MP0250/bortezomib lowers the percentage of idiotype positive cells and the serum levels of M-protein. Overall results define MP0250 as a strong antiangiogenic agent with potential as a novel combination drug for treatment of MM patients.

MP0250, a VEGF and HGF neutralizing DARPin® molecule shows high anti-tumor efficacy in mouse xenograft and patient-derived tumor models.

BACKGROUND: The VEGF/VEGFR and the HGF/cMET pathways are key mediators of the interplay of tumor cells and their microenvironment. However, inhibition of VEGF has been shown to produce only limited clinical benefit and inhibition of the activation of cMET by HGF has not translated into clinical benefit in pivotal trials. MP0250, a DARPin® molecule that specifically inhibits both VEGF and HGF has been developed to explore the clinical potential of dual inhibition of these pathways. RESULTS: MP0250 binding to VEGF and HGF inhibited downstream signalling through VEGFR2 and cMET resulting in inhibition of proliferation of VEGF- and HGF-dependent cells. Antitumor activity was demonstrated in VEGF- and HGF-dependent xenograft and syngeneic models with activity superior to that of individual VEGF- and HGF-blocking DARPin® molecules. Combination therapy studies showed potentiation of the antitumor activity of chemotherapy and immunotherapy agents, including an anti-PD1 antibody. MATERIALS AND METHODS: Potency of MP0250 was assessed in cellular models and in a variety of xenograft models as monotherapy or in combination with standard-of-care drugs. CONCLUSIONS: Dual inhibition of VEGF and HGF by MP0250 produced powerful single agent and combination antitumor activity. This, together with increasing understanding of the role of the HGF/cMET pathway in resistance to VEGF (and other agents), supports testing of MP0250 in the clinic.

Design and characterization of MP0250, a tri-specific anti-HGF/anti-VEGF DARPin® drug candidate.

MP0250 is a multi-domain drug candidate currently being tested in clinical trials for the treatment of cancer. It comprises one anti-vascular endothelial growth factor-A (VEGF-A), one anti-hepatocyte growth factor (HGF), and two anti-human serum albumin (HSA) DARPin® domains within a single polypeptide chain. While there is first clinical validation of a single-domain DARPin® drug candidate, little is known about DARPin® drug candidates comprising multiple domains. Here, we show that MP0250 can be expressed at 15 g/L in soluble form in E. coli high cell-density fermentation, it is stable in soluble/frozen formulation for 2 years as assessed by reverse phase HPLC, it has picomolar potency in inhibiting VEGF-A and HGF in ELISA and cellular assays, and its domains are simultaneously active as shown by surface plasmon resonance. The inclusion of HSA-binding DARPin® domains leads to a favorable pharmacokinetic profile in mouse and cynomolgus monkey, with terminal half-lives of ∼ 30 hours in mouse and ∼ 5 days in cynomolgus monkey. MP0250 is thus a highly potent drug candidate that could be particularly useful in oncology. Beyond MP0250, the properties of MP0250 indicate that multi-domain DARPin® proteins can be valuable next-generation drug candidates.

Novel ubiquitin-derived high affinity binding proteins with tumor targeting properties.

Targeting effector molecules to tumor cells is a promising mode of action for cancer therapy and diagnostics. Binding proteins with high affinity and specificity for a tumor target that carry effector molecules such as toxins, cytokines, or radiolabels to their intended site of action are required for these applications. In order to yield high tumor accumulation while maintaining low levels in healthy tissues and blood, the half-life of such conjugates needs to be in an optimal range. Scaffold-based binding molecules are small proteins with high affinity and short systemic circulation. Due to their low molecular complexity, they are well suited for combination with effector molecules as well as half-life extension technologies yielding therapeutics with half-lives adapted to the specific therapy. We have identified ubiquitin as an ideal scaffold protein due to its outstanding biophysical and biochemical properties. Based on a dimeric ubiquitin library, high affinity and specific binding molecules, so-called Affilin® molecules, have been selected against the extradomain B of fibronectin, a target almost exclusively expressed in tumor tissues. Extradomain B-binding molecules feature high thermal and serum stability as well as strong in vitro target binding and in vivo tumor accumulation. Application of several half-life extension technologies results in molecules of largely unaffected affinity but significantly prolonged in vivo half-life and tumor retention. Our results demonstrate the utility of ubiquitin as a scaffold for the generation of high affinity binders in a modular fashion, which can be combined with effector molecules and half-life extension technologies.

Angiopoietin-2 is critical for cytokine-induced vascular leakage.

Genetic experiments (loss-of-function and gain-of-function) have established the role of Angiopoietin/Tie ligand/receptor tyrosine kinase system as a regulator of vessel maturation and quiescence. Angiopoietin-2 (Ang-2) acts on Tie2-expressing resting endothelial cells as an antagonistic ligand to negatively interfere with the vessel stabilizing effects of constitutive Ang-1/Tie-2 signaling. Ang-2 thereby controls the vascular response to inflammation-inducing as well as angiogenesis-inducing cytokines. This study was aimed at assessing the role of Ang-2 as an autocrine (i.e. endothelial-derived) regulator of rapid vascular responses (within minutes) caused by permeability-inducing agents. Employing two independent in vivo assays to quantitatively assess vascular leakage (tracheal microsphere assay, 1-5 min and Miles assay, 20 min), the immediate vascular response to histamine, bradykinin and VEGF was analyzed in Ang-2-deficient (Ang-2(-/-)) mice. In comparison to the wild type control mice, the Ang2(-/-) mice demonstrated a significantly attenuated response. The Ang-2(-/-) phenotype was rescued by systemic administration (paracrine) of an adenovirus encoding Ang-2. Furthermore, cytokine-induced intracellular calcium influx was impaired in Ang-2(-/-) endothelioma cells, consistent with reduced phospholipase activation in vivo. Additionally, recombinant human Ang-2 (rhAng-2) alone was unable to induce vascular leakage. In summary, we report here in a definite genetic setting that Ang-2 is critical for multiple vascular permeability-inducing cytokines.

Treatment of diabetic macular edema with a designed ankyrin repeat protein that binds vascular endothelial growth factor: a phase I/II study.

PURPOSE: To evaluate the safety and bioactivity of MP0112, a designed ankyrin repeat protein (DARPin) that specifically binds vascular endothelial growth factor (VEGF) in patients with diabetic macular edema (DME). DARPins are a novel class of proteins selected for specific, high-affinity binding to a target protein. DESIGN: Phase I/II, open-label, multicenter dose-escalation trial. METHODS: After a single intravitreal injection of MP0112, the main outcomes were safety assessments, aqueous MP0112 levels, change in best-corrected visual acuity (BCVA), and foveal thickness measured by optical coherence tomography. Six cohorts were planned, but only 3 were enrolled (0.04, 0.15, 0.4 mg), because a maximally tolerated dose of 1.0 mg was identified in a parallel age-related macular degeneration trial. RESULTS: Median aqueous concentration of MP0112 was 555 nM 1 week and >10 nM in 3 of 4 patients 12 weeks post injection of 0.4 mg. Median BCVA improvement at week 12 was 4, 6, and 10 letters in cohorts 1, 2, and 3. Ocular inflammation was observed in 11 patients (61%) and was severe in 1. High-resolution chromatography separated proinflammatory impurities from MP0112, resulting in a new formulation. CONCLUSIONS: A single intraocular injection of 0.4 mg MP0112 resulted in levels above the half-maximal inhibitory concentration and neutralization of VEGF in aqueous humor for 8-12 weeks. Despite inflammation in several patients, there was prolonged edema reduction and improvement in vision in several patients. The source of the inflammation was eliminated from a new preparation that is being tested in an ongoing clinical trial.

Efficacy of sequential treatment with sunitinib-everolimus in an orthotopic mouse model of renal cell carcinoma.

BACKGROUND/AIM: Sequential treatment with targeted agents is standard of care for patients with metastatic renal cell carcinoma (mRCC). However, clinical data directly comparing treatment outcomes with a mammalian target of rapamycin inhibitor or a vascular endothelial growth factor-targeted agent in the second-line setting are lacking. We evaluated sequential treatment in a syngeneic, orthotopic mouse model of mRCC. MATERIALS AND METHODS: BALB/c mice were orthotopically implanted with murine RCC (RENCA) cells expressing luciferase and randomized to vehicle, sunitinib, sunitinib followed by sorafenib, or sunitinib followed by everolimus. Tumor growth and metastases were assessed by in vivo (whole body) and ex vivo (primary tumor, lung, liver) luciferase activity and necropsies, performed on day 20 or 46 for vehicle and treatment groups, respectively. RESULTS: Sunitinib followed by everolimus was associated with reduced luciferase activity and primary tumor weight and volume compared with sunitinib, and sunitinib followed by sorafenib. CONCLUSION: Sequential therapy with sunitinib followed by everolimus demonstrated significant antitumor and anti-metastatic effects.

Angiopoietin-1 mediates inhibition of hypertension-induced release of angiopoietin-2 from endothelial cells.

AIMS: Adequate endothelial cell stimulation is a prerequisite for the adaptive remodelling of macro- and microvessels. A pivotal autocrine mechanism following endothelial cell activation is the release of angiopoietin-2 (Ang-2), which subsequently antagonizes the binding of Ang-1 to the Tie-2 receptor, thus sensitizing the endothelial cells to pro-angiogenic and/or pro-inflammatory stimuli. Based on the observation that hypertension in mice reduces the abundance of Ang-2 stored in arterial endothelial cells, this study was aimed at testing the hypothesis that an increase in wall stress (WS) or stretch-a hallmark of hypertension-is sufficient to release Ang-2 from endothelial cells. METHODS AND RESULTS: In fact, stretching of isolated perfused mouse arteries or human cultured endothelial cells rapidly elicited an increased release of Ang-2. In the cultured endothelial cells, this was preceded by a transient rise in intracellular free calcium, abrogated through calcium chelation and accompanied by a decrease in Tie-2 phosphorylation. Interestingly, Ang-1 abolished the stretch-induced release of Ang-2 from both cultured and native endothelial cells through inhibiting the stretch-dependent mobilization of intracellular calcium. CONCLUSION: Collectively, these results indicate that increased WS or stretch facilitates the release of Ang-2 from endothelial cell Weibel-Palade bodies, and that Ang-1 can block this by attenuating the stretch-mediated rise in intracellular calcium.

Therapeutic interference with EphrinB2 signalling inhibits oxygen-induced angioproliferative retinopathy.

PURPOSE: To investigate whether EphrinB2 (EfnB2) or EphB4 influence retinal angiogenesis under physiological or pathological conditions. METHODS: Using the mouse model of oxygen-induced proliferative retinopathy (OIR), the expression of EfnB2, EphB4, vascular endothelial growth factor (VEGF), VEGFR1 and VEGFR2 was quantified by quantitative polymerase chain reaction (qPCR) and localized in EfnB2- and EphB4-lacZ mice. Angioproliferative retinopathy was manipulated by intravitreal injection of dimeric EfnB2 and monomeric or dimeric EphB4. RESULTS: Dimeric EphB4 (EphB4-Fc) and EfnB2 (EfnB2-Fc) enhanced hypoxia-induced angioproliferative retinopathy but not physiological angiogenesis. Monomeric EphB4 (sEphB4) reduced angiogenesis. The messenger RNA (mRNA) level of EfnB2 increased significantly in the hyperoxic phase (P7-P12), while EphB4, VEGF, VEGFR1 and VEGFR2 showed a significant - up to fivefold - increased expression at P14, the start of morphologically visible vasoproliferation caused by relative hypoxia. CONCLUSION: The ephrin/Eph system is involved in angioproliferative retinopathy. Stimulation of EphB4 and EfnB2 signalling using EfnB2-Fc and EphB4-Fc, respectively, enhanced hypoxia-induced angiogenesis. In contrast, sEphB4 inhibited hypoxia-induced angiogenesis. Therefore, angiogenesis is enhanced by signalling through both EphB4 (forward) and EfnB2 (reverse). The distinction in the expression kinetics of EphB4 and EfnB2 indicates that they govern two different signalling pathways and are regulated in diverse ways. sEphB4 might be a useful drug for antiangiogenic therapy.

Angiopoietin-2 stimulation of endothelial cells induces alphavbeta3 integrin internalization and degradation.

The angiopoietins (Ang-1 and Ang-2) have been identified as agonistic and antagonistic ligands of the endothelial receptor tyrosine kinase Tie2, respectively. Both ligands have been demonstrated to induce translocation of Tie2 to cell-cell junctions. However, only Ang-1 induces Tie2-dependent Akt activation and subsequent survival signaling and endothelial quiescence. Ang-2 interferes negatively with Ang-1/Tie2 signaling, thereby antagonizing the Ang-1/Tie2 axis. Here, we show that both Ang-1 and Ang-2 recruit beta3 integrins to Tie2. This co-localization is most prominent in cell-cell junctions. However, only Ang-2 stimulation resulted in complex formation among Tie2, alphavbeta3 integrin, and focal adhesion kinase as evidenced by co-immunoprecipitation experiments. Focal adhesion kinase was phosphorylated in the FAT domain at Ser(910) upon Ang-2 stimulation and the adaptor proteins p130Cas and talin dissociated from alphavbeta3 integrin. The alphavbeta3 integrin was internalized, ubiquitinylated, and gated toward lysosomes. Taken together, the experiments define Tie2/alphavbeta3 integrin association-induced integrin internalization and degradation as mechanistic consequences of endothelial Ang-2 stimulation.

Workflow comparison for label-free, quantitative secretome proteomics for cancer biomarker discovery: method evaluation, differential analysis, and verification in serum.

The cancer cell secretome has emerged as an attractive subproteome for discovery of candidate blood-based biomarkers. To choose the best performing workflow, we assessed the performance of three first-dimension separation strategies prior to nanoLC-MS/MS analysis: (1) 1D gel electrophoresis (1DGE), (2) peptide SCX chromatography, and (3) tC2 protein reversed phase chromatography. 1DGE using 4-12% gradient gels outperformed the SCX and tC2 methods with respect to number of identified proteins (1092 vs 979 and 580, respectively), reproducibility of protein identification (80% vs 70% and 72%, respectively, assessed in biological N = 3). Reproducibility of protein quantitation based on spectral counting was similar for all 3 methods (CV: 26% vs 24% and 24%, respectively). As a proof-of-concept of secretome proteomics for blood-based biomarker discovery, the gradient 1DGE workflow was subsequently applied to identify IGF1R-signaling related proteins in the secretome of mouse embryonic fibroblasts transformed with human IGF1R (MEF/Toff/IGF1R). VEGF and osteopontin were differentially detected by LC-MS/MS and verified in secretomes by ELISA. Follow-up in serum of mice bearing MEF/Toff/IGF1R-induced tumors showed an increase of osteopontin levels paralleling tumor growth, and reduction in the serum of mice in which IGF1R expression was shut off and tumor regressed.

Affilin molecules selected against the human papillomavirus E7 protein inhibit the proliferation of target cells.

Intracellular binding proteins can be applied as research tools for target validation and study of protein function in cells and potentially as therapeutics. The success of intracellular binding reagents depends on their affinity and specificity for target molecules, although their stability and functionality in the intracellular environment actually determine their usefulness for such application. Alternative binding proteins derived from scaffolds devoid of disulfide bonds are well suited for intracellular use, as their folding and stability are usually not impaired under reducing conditions. Here, we describe the generation of intracellular binding reagents called Affilin, based on the human gammaB-crystallin scaffold. The target was human papillomavirus E7 protein implicated in the development of cervical cancer. E7 binders were selected from the combinatorial gammaB-crystallin library by conventional phage display technique. Affilin variants specifically bound the E7 protein with affinities in the nanomolar range. Intracellular expression of Affilin molecules in E7-positive cells led to inhibition of cellular proliferation. The effect was specific, as the growth of E7-negative cells or cells expressing the wild-type gammaB-crystallin scaffold remained unaffected. These results demonstrate that the gammaB-crystallin scaffold allows the de novo generation of alternative binding proteins, which are suitable for intracellular applications as they retain their functionality in the reducing environment of mammalian cells.

Host-derived angiopoietin-2 affects early stages of tumor development and vessel maturation but is dispensable for later stages of tumor growth.

The angiopoietin/Tie2 system has been identified as the second vascular-specific receptor tyrosine kinase system controlling vessel assembly, maturation, and quiescence. Angiopoietin-2 (Ang-2) is prominently up-regulated in the host-derived vasculature of most tumors, making it an attractive candidate for antiangiogenic intervention. Yet, the net outcome of Ang-2 functions on tumor angiogenesis is believed to be contextual depending on the local cytokine milieu. Correspondingly, Ang-2 manipulatory therapies have been shown to exert protumorigenic as well as antitumorigenic effects. To clarify the role of Ang-2 for angiogenesis and tumor growth in a definite genetic experimental setting, the present study was aimed at comparatively studying the growth of different tumors in wild-type and Ang-2-deficient mice. Lewis lung carcinomas, MT-ret melanomas, and B16F10 melanomas all grew slower in Ang-2-deficient mice. Yet, tumor growth in wild-type and Ang-2-deficient mice dissociated during early stages of tumor development, whereas tumor growth rates during later stages of primary tumor progression were similar. Analysis of the intratumoral vascular architecture revealed no major differences in microvessel density and perfusion characteristics. However, diameters of intratumoral microvessels were smaller in tumors grown in Ang-2-deficient mice, and the vasculature had an altered pattern of pericyte recruitment and maturation. Ang-2-deficient tumor vessels had higher pericyte coverage indices. Recruited pericytes were desmin and NG2 positive and predominately alpha-smooth muscle actin negative, indicative of a more mature pericyte phenotype. Collectively, the experiments define the role of Ang-2 during tumor angiogenesis and establish a better rationale for combination therapies involving Ang-2 manipulatory therapies.

High efficient adenoviral-mediated VEGF and Ang-1 gene delivery into osteogenically differentiated human mesenchymal stem cells.

Survival of ex vivo constructed tissues after transplantation is limited by insufficient oxygen and nutrient supply. Therefore, strategies aiming at improvement of neovascularization of engineered tissues are a key issue in tissue engineering applications. This in vitro study aimed at exploring the usability of osteogenically differentiated human mesenchymal stem cells (MSCs) as carriers of the angiogenic growth factor genes vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) for therapeutic angiogenesis in bone tissue engineering. The ex vivo adenoviral vector mediated transduction into osteogenically differentiated MSCs revealed a highly efficient and long lasting expression of the transgenes. Biological activity of VEGF and Ang-1 secreted from transduced cells was confirmed by analyzing the sprouting, proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs) in response to conditioned medium obtained from transduced cells. The transduced osteogenically differentiated MSCs described in this report may be suitable for inducing neovascularization in bone tissue engineering applications.

Affilin-novel binding molecules based on human gamma-B-crystallin, an all beta-sheet protein.

The concept of novel binding proteins as an alternative to antibodies has undergone rapid development and is now ready for practical use in a wide range of applications. Alternative binding proteins, based on suitable scaffolds with desirable properties, are selected from combinatorial libraries in vitro. Here, we describe an approach using a beta-sheet of human gamma-B-crystallin to generate a universal binding site through randomization of eight solvent-exposed amino acid residues selected according to structural and sequence analyses. Specific variants, so-called Affilin, have been isolated from a phage display library against a variety of targets that differ considerably in size and structure. The isolated Affilin variants can be produced in Escherichia coli as soluble proteins and have a high level of thermodynamic stability. The crystal structures of the human wild-type gamma-B-crystallin and a selected Affilin variant have been determined to 1.7 A and 2.0 A resolution, respectively. Comparison of the two molecules indicates that the human gamma-B-crystallin tolerates amino acid exchanges with no major structural change. We conclude that the intrinsically stable and easily expressed gamma-B-crystallin provides a suitable framework for the generation of novel binding molecules.

Angiopoietins: a link between angiogenesis and inflammation.

The angiopoietin (Ang)-Tie ligand-receptor system has a key regulatory role in regulating vascular integrity and quiescence. Besides its role in angiogenesis, it is an important regulator in numerous diseases including inflammation. Ang-1-mediated Tie2 activation is required to maintain the quiescent resting state of the endothelium. Agonistic Ang-1 functions are antagonized by Ang-2, which is believed to inhibit Ang-1-Tie2 signaling. Ang-2 destabilizes the quiescent endothelium and primes it to respond to exogenous stimuli, thereby facilitating the activities of inflammatory (tumor necrosis factor and interleukin-1) and angiogenic (vascular endothelial growth factor) cytokines. Intriguingly, Ang-2 is expressed weakly by the resting endothelium but becomes strongly upregulated following endothelial activation. Moreover, endothelial cells store Ang-2 in Weibel-Palade bodies from where it can be made available quickly following stimulation, suggesting a role of Ang-2 in controlling rapid vascular adaptive processes. This suggests that Ang-2 is the dynamic regulator of the Ang-Tie2 axis, thereby functioning as a built-in switch controlling the transition of the resting quiescent endothelium towards the activated responsive endothelium.