A 3D tissue model-on-a-chip for studying the effects of human senescent fibroblasts on blood vessels.

All human tissues experience aging that eventually causes organ dysfunction and disease. Cellular senescence was discovered in fibroblasts cultured in vitro. In adults, it is a primary defense mechanism against cancer, but also a major contributor to lifespan limits and disorders associated with aging. To assess how human blood vessels change in an aged environment, we developed an elementary tissue model-on-a-chip that comprises an in vitro three-dimensional model of a blood vessel embedded in a collagen gel with young or senescent skin fibroblasts. We found that senescent fibroblasts mechanically altered the surrounding extracellular matrix by exerting excessive traction stress. We then found that senescent fibroblasts induced sprouting angiogenesis of a microvessel via their senescence-associated secretory phenotype (SASP). Finally, we gathered evidence that the mechanical changes of the microenvironment play a role in sustaining SASP-induced angiogenesis. The model proved useful in monitoring morphological changes in blood vessels induced by senescent fibroblasts while controlling the proportion of senescent cells, and enabled the study of SASP inhibitors, a class of drugs useful in aging and cancer research.

TGF-beta and TNF-alpha cooperatively induce mesenchymal transition of lymphatic endothelial cells via activation of Activin signals.

Lymphatic systems play important roles in the maintenance of fluid homeostasis and undergo anatomical and physiological changes during inflammation and aging. While lymphatic endothelial cells (LECs) undergo mesenchymal transition in response to transforming growth factor-ß (TGF-ß), the molecular mechanisms underlying endothelial-to-mesenchymal transition (EndMT) of LECs remain largely unknown. In this study, we examined the effect of TGF-ß2 and tumor necrosis factor-α (TNF-α), an inflammatory cytokine, on EndMT using human skin-derived lymphatic endothelial cells (HDLECs). TGF-ß2-treated HDLECs showed increased expression of SM22α, a mesenchymal cell marker accompanied by increased cell motility and vascular permeability, suggesting HDLECs to undergo EndMT. Our data also revealed that TNF-α could enhance TGF-ß2-induced EndMT of HDLECs. Furthermore, both cytokines induced the production of Activin A while decreasing the expression of its inhibitory molecule Follistatin, and thus enhancing EndMT. Finally, we demonstrated that human dermal lymphatic vessels underwent EndMT during aging, characterized by double immunostaining for LYVE1 and SM22α. These results suggest that both TGF-ß and TNF-α signals play a central role in EndMT of LECs and could be potential targets for senile edema.

A Fanci knockout mouse model reveals common and distinct functions for FANCI and FANCD2.

Fanconi Anemia (FA) clinical phenotypes are heterogenous and rely on a mutation in one of the 22 FANC genes (FANCA-W) involved in a common interstrand DNA crosslink-repair pathway. A critical step in the activation of FA pathway is the monoubiquitination of FANCD2 and its binding partner FANCI. To better address the clinical phenotype associated with FANCI and the epistatic relationship with FANCD2, we created the first conditional inactivation model for FANCI in mouse. Fanci -/- mice displayed typical FA features such as delayed development in utero, microphtalmia, cellular sensitivity to mitomycin C, occasional limb abnormalities and hematological deficiencies. Interestingly, the deletion of Fanci leads to a strong meiotic phenotype and severe hypogonadism. FANCI was localized in spermatocytes and spermatids and in the nucleus of oocytes. Both FANCI and FANCD2 proteins co-localized with RPA along meiotic chromosomes, albeit at different levels. Consistent with a role in meiotic recombination, FANCI interacted with RAD51 and stimulated D-loop formation, unlike FANCD2. The double knockout Fanci-/- Fancd2-/- also showed epistatic relationship for hematological defects while being not epistatic with respect to generating viable mice in crosses of double heterozygotes. Collectively, this study highlights common and distinct functions of FANCI and FANCD2 during mouse development, meiotic recombination and hematopoiesis.

EGFL7 regulates sprouting angiogenesis and endothelial integrity in a human blood vessel model.

Elucidating the mechanisms underlying sprouting angiogenesis and permeability should enable the development of more effective therapies for various diseases, including retinopathy, cancer, and other vascular disorders. We focused on epidermal growth factor-like domain 7 (EGFL7) which plays an important role in NOTCH signaling and in the organization of angiogenic sprouts. We developed an EGFL7-knockdown in vitro microvessel model and investigated the effect of EGFL7 at a tissue level. We found EGFL7 knockdown suppressed VEGF-A-induced sprouting angiogenesis accompanied by an overproduction of endothelial filopodia and reduced collagen IV deposition at the basal side of endothelial cells. We also observed impaired barrier function which reflected an inflammatory condition. Furthermore, our results showed that proper formation of adherens junctions and phosphorylation of VE-cadherin was disturbed. In conclusion, by using a 3D microvessel model we identified novel roles for EGFL7 in endothelial function during sprouting angiogenesis.

A Vascular Endothelial Growth Factor-Dependent Sprouting Angiogenesis Assay Based on an In Vitro Human Blood Vessel Model for the Study of Anti-Angiogenic Drugs.

Angiogenesis is the formation of new capillaries from pre-existing blood vessels and participates in proper vasculature development. In pathological conditions such as cancer, abnormal angiogenesis takes place. Angiogenesis is primarily carried out by endothelial cells, the innermost layer of blood vessels. The vascular endothelial growth factor-A (VEGF-A) and its receptor-2 (VEGFR-2) trigger most of the mechanisms activating and regulating angiogenesis, and have been the targets for the development of drugs. However, most experimental assays assessing angiogenesis rely on animal models. We report an in vitro model using a microvessel-on-a-chip. It mimics an effective endothelial sprouting angiogenesis event triggered from an initial microvessel using a single angiogenic factor, VEGF-A. The angiogenic sprouting in this model is depends on the Notch signaling, as observed in vivo. This model enables the study of anti-angiogenic drugs which target a specific factor/receptor pathway, as demonstrated by the use of the clinically approved sorafenib and sunitinib for targeting the VEGF-A/VEGFR-2 pathway. Furthermore, this model allows testing simultaneously angiogenesis and permeability. It demonstrates that sorafenib impairs the endothelial barrier function, while sunitinib does not. Such in vitro human model provides a significant complimentary approach to animal models for the development of effective therapies.

A 3D in vitro pericyte-supported microvessel model: visualisation and quantitative characterisation of multistep angiogenesis.

Angiogenesis, which refers to the formation of new blood vessels from already existing vessels, is a promising therapeutic target and a complex multistep process involving many different factors. Pericytes (PCs) are attracting attention as they are considered to make significant contributions to the maturation and stabilisation of newly formed vessels, although not much is known about the precise mechanisms involved. Since there is no single specific marker for pericytes, in vivo models may complicate PC identification and the study of PCs in angiogenesis would benefit from in vitro models recapitulating the interactions between PCs and endothelial cells (ECs) in a three-dimensional (3D) configuration. In this study, a 3D in vitro co-culture microvessel model incorporating ECs and PCs was constructed by bottom-up tissue engineering. Angiogenesis was induced in the manner of sprout formation by the addition of a vascular endothelial cell growth factor. It was found that the incorporation of PCs prevented expansion of the parent vessel diameter and enhanced sprout formation and elongation. Physical interactions between ECs and PCs were visualised by immunostaining and it disclosed that PCs covered the EC monolayer from its basal side in the parent vessel as well as angiogenic sprouts. Furthermore, the microvessels were visualized in 3D by using a non-invasive optical coherence tomography (OCT) imaging system and sprout features were quantitatively assessed. It revealed that the sprouts in EC-PC co-culture vessels were longer and tighter than those in EC mono-culture vessels. The combination of the microvessel model and the OCT system analysis can be useful for the visualisation and demonstration of the multistep process of angiogenesis, which incorporates PCs.

Bundle Gel Fibers with a Tunable Microenvironment for in Vitro Neuron Cell Guiding.

As scaffolds for neuron cell guiding in vitro, gel fibers with a bundle structure, comprising multiple microfibrils, were fabricated using a microfluidic device system by casting a phase-separating polymer blend solution comprising hydroxypropyl cellulose (HPC) and sodium alginate (Na-Alg). The topology and stiffness of the obtained bundle gel fibers depended on their microstructure derived by the polymer blend ratio of HPC and Na-Alg. High concentrations of Na-Alg led to the formation of small microfibrils in a one-bundle gel fiber and stiff characteristics. These bundle gel fibers permitted for the elongation of the neuron cells along their axon orientation with the long axis of fibers. In addition, human-induced pluripotent-stem-cell-derived dopaminergic neuron progenitor cells were differentiated into neuronal cells on the bundle gels. The bundle gel fibers demonstrated an enormous potential as cell culture scaffold materials with an optimal microenvironment for guiding neuron cells.

A Vascular Permeability Assay Using an In Vitro Human Microvessel Model Mimicking the Inflammatory Condition.

The vascular barrier is an important function of the endothelium and its dysfunction is involved in several diseases. The barrier function of the endothelial cell monolayer is governed by cell-cell, cell-extracellular matrix (cell-ECM) contacts, and inflammatory factors such as thrombin, histamine or vascular endothelial growth factor. Several in vivo and in vitro assays that measure the vascular permeability induced by these factors have been developed. However, they suffer limitations such as being challenging for assessing details of biological processes at a cellular level or lacking the architecture of a vessel, that raise the need for new methods. In vitro 3D model-based assays have thus been developed but assays for investigating compounds that protects the barrier function are lacking. Here we describe the development of an in vitro three-dimensional (3D) vascular endothelium model in which we can manipulate the endothelial barrier function and permeability to molecules, which have a molecular weight similar to human serum albumin, allowing to assess the protective effect of compounds. A microvessel was prepared by culturing human umbilical vein endothelial cells (HUVECs) within a collagen gel on a polydimethylsiloxane (PDMS) chip. Using fluorescein isothiocyanate (FITC)-conjugated dextran (70 kDa, FITC-dextran) and confocal fluorescence microscopy, we showed that the microvessel presented an effective barrier function. We were then able to induce the loss of this barrier function by treatment with the inflammatory factor thrombin. The loss of barrier function was quantified by the extravasation of FITC-dextran into collagen matrix. Furthermore, we were able to analyze the protective effect on the endothelial barrier function of the cyclic adenosine monophosphate (cAMP) analog, 8-pCPT-2'-O-Me-cAMP (also called 007). In an attempt to understand the effects of thrombin and 007 in our model, we analyzed the adherens junctions and cytoskeleton through immunostaining of the vascular endothelial cadherin and actin, respectively. Our assay method could be used to screen for compounds modulating the barrier function of endothelial cells, as well as investigating mechanistic aspects of barrier dysfunction.

Cancer-causing mutations in the tumor suppressor PALB2 reveal a novel cancer mechanism using a hidden nuclear export signal in the WD40 repeat motif.

One typical mechanism to promote genomic instability, a hallmark of cancer, is to inactivate tumor suppressors, such as PALB2. It has recently been reported that mutations in PALB2 increase the risk of breast cancer by 8-9-fold by age 40 and the life time risk is ∼3-4-fold. To date, predicting the functional consequences of PALB2 mutations has been challenging as they lead to different cancer risks. Here, we performed a structure-function analysis of PALB2, using PALB2 truncated mutants (R170fs, L531fs, Q775X and W1038X), and uncovered a new mechanism by which cancer cells could drive genomic instability. Remarkably, the PALB2 W1038X mutant, harboring a mutation in its C-terminal domain, is still proficient in stimulating RAD51-mediated recombination in vitro, although it is unusually localized to the cytoplasm. After further investigation, we identified a hidden NES within the WD40 domain of PALB2 and found that the W1038X truncation leads to the exposure of this NES to CRM1, an export protein. This concept was also confirmed with another WD40-containing protein, RBBP4. Consequently, our studies reveal an unreported mechanism linking the nucleocytoplasmic translocation of PALB2 mutants to cancer formation.

Roles for APRIN (PDS5B) in homologous recombination and in ovarian cancer prediction.

APRIN (PDS5 cohesin associated factor B) interacts with both the cohesin complex and the BRCA2 tumor suppressor. How APRIN influences cohesion and DNA repair processes is not well understood. Here, we show that APRIN is recruited to DNA damage sites. We find that APRIN interacts directly with RAD51, PALB2 and BRCA2. APRIN stimulates RAD51-mediated DNA strand invasion. APRIN also binds DNA with an affinity for D-loop structures and single-strand (ss) DNA. APRIN is a new homologous recombination (HR) mediator as it counteracts the RPA inhibitory effect on RAD51 loading to ssDNA. We show that APRIN strongly improves the annealing of complementary-strand DNA and that it can stimulate this process in synergy with BRCA2. Unlike cohesin constituents, its depletion has no impact on class switch recombination, supporting a specific role for this protein in HR. Furthermore, we show that low APRIN expression levels correlate with a better survival in ovarian cancer patients and that APRIN depletion sensitizes cells to the PARP inhibitor Olaparib in xenografted zebrafish. Our findings establish APRIN as an important and specific actor of HR, with cohesin-independent functions.

Investigation of the DNA damage response to SFOM-0046, a new small-molecule drug inducing DNA double-strand breaks.

2-Ethylphenyl 4-(3-ethylureido)benzenesulfonate (SFOM-0046) is a novel anticancer agent that arrests cell cycle in S-phase and causes DNA replication stress leading to the phosphorylation of H2AX into γ-H2AX. First, using the M21, HT29, HT-1080 and HeLa cell lines, we confirmed that S-phase cell cycle arrest and γ-H2AX foci induction by SFOM-0046 is a general mechanism occurring in diverse cancer cell lines. In addition to γ-H2AX, SFOM-0046 activates preferentially ATR-Chk1 in M21 and HT29 cells while both ATR-Chk1 and ATM-Chk2 pathways are activated in HCT116 cells. Co-localization of SFOM-0046-induced 53BP1 foci with γ-H2AX foci validates that the DNA damage generated corresponds to double-strand-breaks (DSBs). Consistent with an S-phase arrest, SFOM-0046 treatment induces RAD51 foci formation but not DNA-PKcs foci, confirming that homologous recombination is the major DSB repair pathway targeted by the drug. Furthermore, using isogenic HCT116 p53+/+ and HCT116 p53-/- cells, we showed that p53 plays a key role in the survival mechanism to SFOM-0046. Finally, SFOM-0046 exhibits a dose-dependent antitumor activity on human fibrosarcoma HT-1080 tumours grafted onto chick chorioallantoic membranes without showing embryo toxicity even at high doses. Altogether, our results highlight SFOM-0046 as a very promising drug that induces a replication stress response.

Exploring the roles of PALB2 at the crossroads of DNA repair and cancer.

PALB2 [partner and localizer of BRCA2 (breast cancer early-onset 2)] [corrected] has emerged as a key player in the maintenance of genome integrity. Biallelic mutations in PALB2 cause FA (Fanconi's anaemia) subtype FA-N, a devastating inherited disorder marked by developmental abnormalities, bone marrow failure and childhood cancer susceptibility, whereas monoallelic mutations predispose to breast, ovarian and pancreatic cancer. The tumour suppressor role of PALB2 has been intimately linked to its ability to promote HR (homologous recombination)-mediated repair of DNA double-strand breaks. Because PALB2 lies at the crossroads between FA, HR and cancer susceptibility, understanding its function has become the primary focus of several studies. The present review discusses a current synthesis of the contribution of PALB2 to these pathways. We also provide a molecular description of FA- or cancer-associated PALB2 mutations.

Breast cancer proteins PALB2 and BRCA2 stimulate polymerase η in recombination-associated DNA synthesis at blocked replication forks.

One envisioned function of homologous recombination (HR) is to find a template for DNA synthesis from the resected 3'-OH molecules that occur during double-strand break (DSB) repair at collapsed replication forks. However, the interplay between DNA synthesis and HR remains poorly understood in higher eukaryotic cells. Here, we reveal functions for the breast cancer proteins BRCA2 and PALB2 at blocked replication forks and show a role for these proteins in stimulating polymerase η (Polη) to initiate DNA synthesis. PALB2, BRCA2, and Polη colocalize at stalled or collapsed replication forks after hydroxyurea treatment. Moreover, PALB2 and BRCA2 interact with Polη and are required to sustain the recruitment of Polη at blocked replication forks. PALB2 and BRCA2 stimulate Polη-dependent DNA synthesis on D loop substrates. We conclude that PALB2 and BRCA2, in addition to their functions in D loop formation, play crucial roles in the initiation of recombination-associated DNA synthesis by Polη-mediated DNA repair.

GST-His purification: a two-step affinity purification protocol yielding full-length purified proteins.

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST) and a C-terminal 10xHis tag, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.