High-content image analysis to study phenotypic heterogeneity in endothelial cell monolayers.

Endothelial cells (ECs) are heterogeneous across and within tissues, reflecting distinct, specialised functions. EC heterogeneity has been proposed to underpin EC plasticity independently from vessel microenvironments. However, heterogeneity driven by contact-dependent or short-range cell-cell crosstalk cannot be evaluated with single cell transcriptomic approaches, as spatial and contextual information is lost. Nonetheless, quantification of EC heterogeneity and understanding of its molecular drivers is key to developing novel therapeutics for cancer, cardiovascular diseases and for revascularisation in regenerative medicine. Here, we developed an EC profiling tool (ECPT) to examine individual cells within intact monolayers. We used ECPT to characterise different phenotypes in arterial, venous and microvascular EC populations. In line with other studies, we measured heterogeneity in terms of cell cycle, proliferation, and junction organisation. ECPT uncovered a previously under-appreciated single-cell heterogeneity in NOTCH activation. We correlated cell proliferation with different NOTCH activation states at the single-cell and population levels. The positional and relational information extracted with our novel approach is key to elucidating the molecular mechanisms underpinning EC heterogeneity.

The impact of intensity-modulated radiation treatment on dento-alveolar microvasculature in pharyngeal cancer implant patients.

OBJECTIVES: Dental rehabilitation post-radiotherapy often requires the consideration of dental implants. However, these are tentatively prescribed due to the concern of hypovascularisation and possible osteoradionecrosis. Hence, the current study assessed the microvasculature of the dento-alveolar bone at implant sites taking into consideration the exact radiotherapy dose received to the region. MATERIALS AND METHODS: Bone cores were taken from nine patients during implant treatment and compared to nine control patients. Specimens were stained using CD31 and digitalised using a high-resolution scanner for qualitative and quantitative assessment of the microvasculature. Monaco® treatment planning system was used to volume the implant site providing mean dose (Dmean ) and maximum dose (Dmax ). RESULTS: A total of 23 bone cores were retrieved for analysis. The cohort had a Dmean of 38.4 Gy (59.6-24.3 Gy). Qualitative analysis identified a clear reduction in the miniscule terminal capillaries and high incidence of obliterated lumens with increasing radiotherapy. Microvasculature density of irradiated patients was markedly reduced (P = .0034) compared to the control group with an inverse correlation to RT doses (P < .0001). Specifically, doses up to 30 Gy appear to preserve sufficient vascularisation (~77% in comparison with control) and tissue architecture. By contrast, exposure to higher doses 40%-61% of the micro-vessels were lost. CONCLUSION: Intensity-modulated radiotherapy doses above 30 Gy identified reduction in microvasculature which is a lower threshold than previously accepted. In pharyngeal cancer patients' doses to the jaw bones often exceed this threshold. Coupled with favourable survival in certain oropharyngeal and nasopharyngeal cancer, dental rehabilitation via implants provides a significant clinical challenge.

Angiopoietin-2 in Bone Marrow milieu promotes Multiple Myeloma-associated angiogenesis.

Angiopoietin-2 (Ang-2) is involved in angiogenesis in both solid and hematological malignancies. In Multiple Myeloma (MM), serum Ang-2 correlates with disease progression and response to therapy. To address the patho-physiologic role of Ang-2 in MM associated angiogenesis, we used sera from patients with active MM, which contained significantly higher levels of the molecule, compared to those from patients with smoldering MM and Monoclonal Gammopathy of Undetermined Significance. MM Bone Marrow (BM) sera with high Ang-2 concentration specifically contributed to endothelial cell (EC) activation, while Ang-1 containing sera maintained EC stabilization. The functional dichotomy of Ang-1 and Ang-2 was confirmed by the triggering of distinctive signaling pathways down-stream the common Tie-2 receptor, i.e., the Akt or the ERK- phosphorylation pathway. Notably, Ang-2 but not VEGF serum levels correlated with BM micro-vessel density, further underscoring the key role of Ang-2 in angiogenesis. Western Blot, RT-PCR and immunocytochemistry identified MMEC as the major source of Ang-2, at variance with MM cells and CD14(+) BM monocytes. These data suggest that Ang-2 produced in the BM milieu may contribute to MM angiogenesis and suggest that the molecule can be further exploited both as angiogenesis biomarker and as a potential therapeutic target.

Multifactorial estimation of clinical outcome in HPV-associated oropharyngeal squamous cell carcinoma via automated image analysis of routine diagnostic H&E slides and neural network modelling.

OBJECTIVE: Routine haematoxylin and eosin (H&E) photomicrographs from human papillomavirus-associated oropharyngeal squamous cell carcinomas (HPV + OpSCC) contain a wealth of prognostic information. In this study, we developed a high content image analysis (HCIA) workflow to quantify features of H&E images from HPV + OpSCC patients to identify prognostic features and predict patient outcomes. METHODS: First, we have developed an open-source HCIA tool for single-cell segmentation and classification of H&E images. Subsequently, we have used our HCIA tool to analyse a set of 889 images from diagnostic H&E slides in a retrospective cohort of HPV + OpSCC patients with favourable (FO, n = 60) or unfavourable (UO, n = 30) outcomes. We have identified and measured 31 prognostic features which were quantified in each sample and used to train a neural network (NN) model to predict patient outcomes. RESULTS: Univariate and multivariate statistical analyses revealed significant differences between FO and UO patients in 31 and 17 variables, respectively (P < 0.05). At the single-image level, the NN model had an overall accuracy of 72.5% and 71.2% in recognising FO and UO patients when applied to test or validation sets, respectively. When considering 10 images per patient, the accuracy of the NN model increased to 86.7% in the test set. CONCLUSION: Our open-source H&E analysis workflow and predictive models confirm previously reported prognostic features and identifies novel factors which predict HPV + OpSCC outcomes with promising accuracy. Our work supports the use of machine learning in digital pathology to exploit clinically relevant features in routine diagnostic pathology without additional biomarkers.

Continuously perfusable, customisable, and matrix-free vasculature on a chip platform.

Creating vascularised cellular environments in vitro is a current challenge in tissue engineering and a bottleneck towards developing functional stem cell-derived microtissues for regenerative medicine and basic investigations. Here we have developed a new workflow to manufacture vasculature on chip (VoC) systems efficiently, quickly, and inexpensively. We have employed 3D printing for fast-prototyping of bespoke VoC and coupled them with a refined organotypic culture system (OVAA) to grow patent capillaries in vitro using tissue-specific endothelial and stromal cells. Furthermore, we have designed and implemented a pocket-size flow driver to establish physiologic perfusive flow throughout our VoC-OVAA with minimal medium use and waste. Using our platform, we have created vascularised microtissues and perfused them at physiologic flow rates for extended time (>2 weeks) observing flow-dependent vascular remodelling. Overall, we present for the first time a scalable and customisable system to grow vascularised and perfusable microtissues, a key initial step to grow mature and functional tissues in vitro. We envision that this technology will empower fast prototyping and validation of increasingly biomimetic in vitro systems, including interconnected multi-tissue systems.

The vasostatin-1 fragment of chromogranin A preserves a quiescent phenotype in hypoxia-driven endothelial cells and regulates tumor neovascularization.

The angiogenic switch is a fundamental process for many diseases and for tumor growth. The main proangiogenic stimulus is hypoxia, through activation of the hypoxia-inducible factor (HIF)-1α pathway in endothelial cells (ECs). We have previously shown that the vasostatin-1 (VS-1) fragment of chromogranin A inhibits TNF-α-induced vessel permeability and VEGF-induced EC proliferation, together with migration and matrix invasion, which are all critical steps in angiogenesis. The present study was undertaken to investigate the effect of VS-1 on tumor angiogenesis. We found mouse mammary adenocarcinomas (TS/A), genetically engineered to secrete VS-1 (TS/A 1B8), to be characterized by reduced vascular density and more regular vessels, compared with nontransfected tumors [TS/A wild type (WT)]. Supernatants from TS/A WT cells, but not those from TS/A 1B8, generated tip cells and promoted the permeability of primary human umbilical vein ECs, via VE-cadherin redistribution and cytoskeletal disorganization. These effects were abrogated by mAb 5A8, a VS-1-blocking antibody. Furthermore, VS-1 inhibited hypoxia-driven EC morphological changes, VE-cadherin redistribution, intercellular gap formation, tube morphogenesis, and HIF-1α nuclear translocation in vitro. Our findings highlight a previously undescribed function of VS-1 as a regulator of tumor vascularization.

Assessing biocompatibility & mechanical testing of 3D-printed PEEK versus milled PEEK.

Objectives: To compare mechanical properties of 3D-printed and milled poly-ether-ether-ketone (PEEK) materials. To define post-production treatments to enhance biocompatibility of 3D-printed PEEK. Methods: Standardised PEEK samples were produced via milling and fused-deposition-modelling 3D-printing. To evaluate mechanical properties, tensile strength, maximum flexural strength, fracture toughness, and micro-hardness were measured.3D printed samples were sandblasted with 50 or 125 µm aluminium oxide beads to increase biocompatibility.Scanning electron microscopy (SEM) evaluated microstructure of 3D-printed and sandblasted samples, estimating surface roughness at scales from 1mm-1µm.Cell adhesion on 3D printed and sandblasted materials was evaluated by culturing primary human endothelial cells and osteoblasts (HUVEC, HOBS) and evaluating cell growth over 48 h. Results: 3D printed materials had lower tensile strength, flexural strength, and fracture toughness, but higher micro-hardness.SEM analysis of 3D-printed surfaces showed sandblasting with 125 and 50 µm silica particles removed printing defects and created roughened surfaces for increased HUVEC and HOBs uniform cell adhesion and distribution. No cytotoxicity was observed over a 48h period, and all cells demonstrated >95% viability. Clinical significance: 3D-printing of PEEK is an emerging technology with clear advantages over milling in maxillofacial implant production. Nonetheless, this manufacturing modality may produce 3D printed PEEK devices with lower mechanical resistance parameters compared to milled PEEK but with values compatible with natural bone. PEEK has poor osteoconductivity and ability to osseointegrate. Sandblasting is an inexpensive modality to remove irregular surface defects and create uniform micro-rough surfaces supporting cell attachment and potentially enhancing integration of PEEK implants with host tissue.

3D Models as a Tool to Assess the Anti-Tumor Efficacy of Therapeutic Antibodies: Advantages and Limitations.

Therapeutic monoclonal antibodies (mAbs) are an emerging and very active frontier in clinical oncology, with hundred molecules currently in use or being tested. These treatments have already revolutionized clinical outcomes in both solid and hematological malignancies. However, identifying patients who are most likely to benefit from mAbs treatment is currently challenging and limiting the impact of such therapies. To overcome this issue, and to fulfill the expectations of mAbs therapies, it is urgently required to develop proper culture models capable of faithfully reproducing the interactions between tumor and its surrounding native microenvironment (TME). Three-dimensional (3D) models which allow the assessment of the impact of drugs on tumors within its TME in a patient-specific context are promising avenues to progressively fill the gap between conventional 2D cultures and animal models, substantially contributing to the achievement of personalized medicine. This review aims to give a brief overview of the currently available 3D models, together with their specific exploitation for therapeutic mAbs testing, underlying advantages and current limitations to a broader use in preclinical oncology.

DAR 16-II Primes Endothelial Cells for Angiogenesis Improving Bone Ingrowth in 3D-Printed BCP Scaffolds and Regeneration of Critically Sized Bone Defects.

Bone is a highly vascularized tissue and relies on the angiogenesis and response of cells in the immediate environmental niche at the defect site for regeneration. Hence, the ability to control angiogenesis and cellular responses during osteogenesis has important implications in tissue-engineered strategies. Self-assembling ionic-complementary peptides have received much interest as they mimic the natural extracellular matrix. Three-dimensional (3D)-printed biphasic calcium phosphate (BCP) scaffolds coated with self-assembling DAR 16-II peptide provide a support template with the ability to recruit and enhance the adhesion of cells. In vitro studies demonstrated prompt the adhesion of both human umbilical vein endothelial cells (HUVEC) and human mesenchymal stem cells (hMSC), favoring endothelial cell activation toward an angiogenic phenotype. The SEM-EDS and protein micro bicinchoninic acid (BCA) assays demonstrated the efficacy of the coating. Whole proteomic analysis of DAR 16-II-treated HUVECs demonstrated the upregulation of proteins involved in cell adhesion (HABP2), migration (AMOTL1), cytoskeletal re-arrangement (SHC1, TMOD2), immuno-modulation (AMBP, MIF), and morphogenesis (COL4A1). In vivo studies using DAR-16-II-coated scaffolds provided an architectural template, promoting cell colonization, osteogenesis, and angiogenesis. In conclusion, DAR 16-II acts as a proactive angiogenic factor when adsorbed onto BCP scaffolds and provides a simple and effective functionalization step to facilitate the translation of tailored 3D-printed BCP scaffolds for clinical applications.

Bortezomib induces autophagic death in proliferating human endothelial cells.

The proteasome inhibitor Bortezomib has been approved for the treatment of relapsed/refractory multiple myeloma (MM), thanks to its ability to induce MM cell apoptosis. Moreover, Bortezomib has antiangiogenic properties. We report that endothelial cells (EC) exposed to Bortezomib undergo death to an extent that depends strictly on their activation state. Indeed, while quiescent EC are resistant to Bortezomib, the drug results maximally toxic in EC switched toward angiogenesis with FGF, and exerts a moderate effect on subconfluent HUVEC. Moreover, EC activation state deeply influences the death pathway elicited by Bortezomib: after treatment, angiogenesis-triggered EC display typical features of apoptosis. Conversely, death of subconfluent EC is preceded by ROS generation and signs typical of autophagy, including intense cytoplasmic vacuolization with evidence of autophagosomes at electron microscopy, and conversion of the cytosolic MAP LC3 I form toward the autophagosome-associated LC3 II form. Treatment with the specific autophagy inhibitor 3-MA prevents both LC3 I/LC3 II conversion and HUVEC cell death. Finally, early removal of Bortezomib is accompanied by the recovery of cell shape and viability. These findings strongly suggest that Bortezomib induces either apoptosis or autophagy in EC; interfering with the autophagic response may potentiate the antiangiogenic effect of the drug.

High-Content Imaging to Phenotype Human Primary and iPSC-Derived Cells.

Increasingly powerful microscopy, liquid handling, and computational techniques have enabled cell imaging in high throughput. Microscopy images are quantified using high-content analysis platforms linking object features to cell behavior. This can be attempted on physiologically relevant cell models, including stem cells and primary cells, in complex environments, and conceivably in the presence of perturbations. Recently, substantial focus has been devoted to cell profiling for cell therapy, assays for drug discovery or biomarker identification for clinical decision-making protocols, bringing this wealth of information into translational applications. In this chapter, we focus on two protocols enabling to (1) benchmark human cells, in particular human endothelial cells as a case study and (2) extract cells from blood for follow-up experiments including image-based drug testing. We also present concepts of high-content imaging and discuss the benefits and challenges, with the aim of enabling readers to tailor existing pipelines and bring such approaches closer to translational research and the clinic.

Biofunctionalization of bioactive ceramic scaffolds to increase the cell response for bone regeneration.

Biofunctionalization was investigated for polymers and metals considering their scarce integration ability. On the contrary few studies dealt with ceramic biofunctionalization because the bioactive and bioresorbable surfaces of ceramics are able to positively interact with biological environment. In this study the cell-response improvement on biofunctionalized wollastonite and diopside-based scaffolds was demonstrated. The ceramics were first obtained by heat treatment of a silicone embedding reactive oxide fillers and then biofunctionalized with adhesive peptides mapped on vitronectin. The most promisingin vitroresults, in terms of h-osteoblast proliferation and bone-related gene expression, were reached anchoring selectively a peptide stable toward proteolytic degradation induced by serum-enriched medium. Inin vivoassays the anchoring of this protease-stable adhesive peptide was combined with self-assembling peptides, for increasing cell viability and angiogenesis. The results demonstrated external and internal cell colonization of biofunctionalized scaffolds with formation of new blood vessels (neoangiogenesis) and stimulation of ectopic mineralization.

Increased expression and secretion of resistin in epicardial adipose tissue of patients with acute coronary syndrome.

The purpose of this study was to test the hypothesis that specific epicardial adipose tissue (EAT) proinflammatory adipokines might be implicated in acute coronary syndrome (ACS). We compared expression and protein secretion of several EAT adipokines of male ACS with those of matched stable coronary artery disease (CAD) patients and controls with angiographically normal coronary arteries. The effect of supernatant of cultured EAT on endothelial cell permeability in vitro was also evaluated in the three study groups. EAT of ACS patients showed significantly higher gene expression and protein secretion of resistin than patients with stable CAD. Interleukin-6, plasminogen activator inhibitor-1, and monocyte chemoattractant protein-1 genes were also significantly overexpressed in ACS compared with the control group but not when compared with stable CAD. Immunofluorescence of EAT sections revealed a significantly greater number of CD68(+) cells in ACS patients than stable CAD and control groups. The permeability of endothelial cells in vitro was significantly increased after exposure to supernatant of cultured EAT from ACS, but not control or stable CAD groups, and this effect was normalized by anti-resistin antiserum. We found that EAT of patients with ACS is characterized by increased expression and secretion of resistin and associated with increased in vitro endothelial cell permeability.

Biomimetic gradient scaffold of collagen-hydroxyapatite for osteochondral regeneration.

Osteochondral defects remain a major clinical challenge mainly due to the combined damage to the articular cartilage and the underlying bone, and the interface between the two tissues having very different properties. Current treatment modalities have several limitations and drawbacks, with limited capacity of restoration; however, tissue engineering shows promise in improving the clinical outcomes of osteochondral defects. In this study, a novel gradient scaffold has been fabricated, implementing a gradient structure in the design to mimic the anatomical, biological and physicochemical properties of bone and cartilage as closely as possible. Compared with the commonly studied multi-layer scaffolds, the gradient scaffold has the potential to induce a smooth transition between cartilage and bone and avoid any instability at the interface, mimicking the natural structure of the osteochondral tissue. The scaffold comprises a collagen matrix with a gradient distribution of low-crystalline hydroxyapatite particles. Physicochemical analyses confirmed phase and chemical compositions of the gradient scaffold and the distribution of the mineral phase along the gradient scaffold. Mechanical tests confirmed the gradient of stiffness throughout the scaffold, according to its mineral content. The gradient scaffold exhibited good biological performances both in vitro and in vivo. Biological evaluation of the scaffold, in combination with human bone-marrow-derived mesenchymal stem cells, demonstrated that the gradient of composition and stiffness preferentially increased cell proliferation in different sub-regions of the scaffold, according to their high chondrogenic or osteogenic characteristics. The in vivo biocompatibility of the gradient scaffold was confirmed by its subcutaneous implantation in rats. The gradient scaffold was significantly colonised by host cells and minimal foreign body reaction was observed. The scaffold's favourable chemical, physical and biological properties demonstrated that it has good potential as an engineered osteochondral analogue for the regeneration of damaged tissue.

Integrated Multiparametric High-Content Profiling of Endothelial Cells.

Endothelial cells (ECs) are widely heterogeneous at the cell level and serve different functions at the vessel and tissue levels. EC-forming colonies derived from induced pluripotent stem cells (iPSC-ECFCs) alongside models such as primary human umbilical vein ECs (HUVECs) are slowly becoming available for research with future applications in cell therapies, disease modeling, and drug discovery. We and others previously described high-content analysis approaches capturing unbiased morphology-based measurements coupled with immunofluorescence and used these for multidimensional reduction and population analysis. Here, we report a tailored workflow to characterize ECs. We acquire images at high resolution with high-magnification water-immersion objectives with Hoechst, vascular endothelial cadherin (VEC), and activated NOTCH staining. We hypothesize that via these key markers alone we would be able to distinguish and assess different EC populations. We used cell population software analysis to phenotype HUVECs and iPSC-ECFCs in the absence or presence of vascular endothelial growth factor (VEGF). To our knowledge, this study presents the first parallel quantitative high-content multiparametric profiling of EC models. Importantly, it highlights a simple strategy to benchmark ECs in different conditions and develop new approaches for biological research and translational applications for regenerative medicine.

The vasostatin-I fragment of chromogranin A inhibits VEGF-induced endothelial cell proliferation and migration.

UNLABELLED: A growing body of evidence suggests that chromogranin A (CgA), a secretory protein released by many neuroendocrine cells and frequently used as a diagnostic and prognostic serum marker for a range of neuroendocrine tumors, is a precursor of several bioactive fragments. This work was undertaken to assess whether the N-terminal fragment CgA(1-76) (called vasostatin I) can inhibit the proangiogenic activity of vascular endothelial growth factor (VEGF), a factor involved in tumor growth. The effect of recombinant human vasostatin I (VS-1) on VEGF-induced human umbilical endothelial cells (HUVEC) signaling, proliferation, migration, and organization has been investigated. We have found that VS-1 (3 microg/ml; 330 nM) can inhibit VEGF-induced ERK phosphorylation, as well as cell migration, proliferation, morphogenesis, and invasion of collagen gels in various in vitro assays. In addition, VS-1 could inhibit the formation of capillary-like structures in Matrigel plugs in a rat model. VS-1 could also inhibit basal ERK phosphorylation and motility of HUVEC, leading to a more quiescent state in the absence of VEGF, without inducing apoptotic or necrotic effects. CONCLUSION: These findings suggest that vasostatin I may play a novel role as a regulator of endothelial cell function and homeostasis.

Protective effect of TAT-delivered alpha-synuclein: relevance of the C-terminal domain and involvement of HSP70.

Alpha-synuclein (alpha-syn) is a 140-amino acid presinaptic protein whose mutations A30P and A53T have been linked to familiar Parkinson's disease (PD). Many data suggest that alpha-syn aggregation is the key event that triggers alpha-syn-mediated neurotoxicity. Nevertheless, other lines of evidence proposed a protective role of alpha-syn against oxidative stress (a major feature of PD), even if the exact mechanism of this protective action and the role of the pathogenetic mutations to this respect have not been elucidated yet. To address these points, we developed an in vitro model of oxidative stress by exposing PC12 cells to hydrogen peroxide (H2O2) (150 microM) for 72 h, and we evaluated alpha-syn-mediated protection delivering increasing amounts of alpha-syn (wild type [WT] or mutated) inside cells using the fusion proteins TAT-alpha-syn (WT, A30P, and A53T). We found that nanomolar amounts of TAT-alpha-syn-mediated protected against oxidative stress and other cellular injuries (6-hydroxydopamine and serum deprivation), whereas micromolar amounts of the fusion proteins were intrinsically toxic to cells. The protective effect was independent from the presence of the mutations A30P and A53T, but no protection occurred when cells were challenged with the proteasome inhibitors lactacystin and MG132. We verified that the protection mechanism required the presence of the C-terminal domain of alpha-syn, as nanomolar amounts of the C-terminal truncated fusion protein TAT-alpha-syn (WT[1-97]) failed in preventing H2O2 toxicity. To further characterize the molecular mechanisms at the basis of alpha-syn protection, we investigated the possible involvement of the chaperone protein HSP70 that is widely implicated in neuroprotection. We found that, at nanomolar concentrations, TAT-alpha-syn was able to increase HSP70 protein level, whereas at the micromolar scale, TAT-alpha-syn decreased HSP70 at the protein level. These effects on HSP70 were independent from the presence of alpha-syn pathogenetic mutations but required the alpha-syn C-terminal domain. The implications for alpha-syn-mediated neurotoxicity and for PD pathogenesis and progression are discussed.

PHD2 regulates arteriogenic macrophages through TIE2 signalling.

Occlusion of the main arterial route redirects blood flow to the collateral circulation. We previously reported that macrophages genetically modified to express low levels of prolyl hydroxylase domain protein 2 (PHD2) display an arteriogenic phenotype, which promotes the formation of collateral vessels and protects the skeletal muscle from ischaemic necrosis. However, the molecular mechanisms underlying this process are unknown. Here, we demonstrate that femoral artery occlusion induces a switch in macrophage phenotype through angiopoietin-1 (ANG1)-mediated Phd2 repression. ANG blockade by a soluble trap prevented the downregulation of Phd2 expression in macrophages and their phenotypic switch, thus inhibiting collateral growth. ANG1-dependent Phd2 repression initiated a feed-forward loop mediated by the induction of the ANG receptor TIE2 in macrophages. Gene silencing and cell depletion strategies demonstrate that TIE2 induction in macrophages is required to promote their proarteriogenic functions, enabling collateral vessel formation following arterial obstruction. These results indicate an indispensable role for TIE2 in sustaining in situ programming of macrophages to a proarteriogenic, M2-like phenotype, suggesting possible new venues for the treatment of ischaemic disorders.

Gene-targeting of Phd2 improves tumor response to chemotherapy and prevents side-toxicity.

The success of chemotherapy in cancer treatment is limited by scarce drug delivery to the tumor and severe side-toxicity. Prolyl hydroxylase domain protein 2 (PHD2) is an oxygen/redox-sensitive enzyme that induces cellular adaptations to stress conditions. Reduced activity of PHD2 in endothelial cells normalizes tumor vessels and enhances perfusion. Here, we show that tumor vessel normalization by genetic inactivation of Phd2 increases the delivery of chemotherapeutics to the tumor and, hence, their antitumor and antimetastatic effect, regardless of combined inhibition of Phd2 in cancer cells. In response to chemotherapy-induced oxidative stress, pharmacological inhibition or genetic inactivation of Phd2 enhances a hypoxia-inducible transcription factor (HIF)-mediated detoxification program in healthy organs, which prevents oxidative damage, organ failure, and tissue demise. Altogether, our study discloses alternative strategies for chemotherapy optimization.

Hypoxia-inducible transcription factor-1 alpha determines sensitivity of endothelial cells to the proteosome inhibitor bortezomib.

Angiogenesis is a complex, orchestrated process that plays a critical role in several conditions and has special relevance in the progression of cancer. Hypoxia is the major stimulus for angiogenesis, and hypoxia-inducible transcription factor-1 alpha (HIF-1alpha) is its key mediator. We set up a novel in vitro model of HIF-1alpha up-regulation by treating human umbilical vein endothelial cells (HUVECs) with the hypoxia-mimicking deferoxamine (DFO) and found that this condition was sufficient to promote angiogenesis, like the well-known HUVEC model cultured under low pO(2.) The proteasome inhibitor bortezomib, which induces strong apoptosis in cancer cells, abrogated proliferation and angiogenesis of HUVECs when used at a high concentration (100 nM), yet promoted both functions at a low dosage (10 nM). This double-edged effect appeared to be mediated by differential effects exerted by the different concentrations of bortezomib on 2 master regulators of tumor-associated angiogenesis, HIF-1alpha and nuclear factor kappa B (NF-kB). Significantly, when HUVECs were induced to express HIF-1alpha prior to bortezomib treatment, proliferative and angiogenic responses were abolished, and a greatly enhanced proapoptotic effect was promoted with both concentrations of the drug. These findings indicate that HIF-1alpha up-regulation may sensitize endothelial cells to the antiangiogenic and proapoptotic effects of bortezomib and might be exploited to target tumor-associated vessels in the course of antiangiogenic therapies.