Magnetic resonance imaging and ultrasound elastography in the context of preclinical pharmacological research: significance for the 3R principles.

The 3Rs principles-reduction, refinement, replacement-are at the core of preclinical research within drug discovery, which still relies to a great extent on the availability of models of disease in animals. Minimizing their distress, reducing their number as well as searching for means to replace them in experimental studies are constant objectives in this area. Due to its non-invasive character in vivo imaging supports these efforts by enabling repeated longitudinal assessments in each animal which serves as its own control, thereby enabling to reduce considerably the animal utilization in the experiments. The repetitive monitoring of pathology progression and the effects of therapy becomes feasible by assessment of quantitative biomarkers. Moreover, imaging has translational prospects by facilitating the comparison of studies performed in small rodents and humans. Also, learnings from the clinic may be potentially back-translated to preclinical settings and therefore contribute to refining animal investigations. By concentrating on activities around the application of magnetic resonance imaging (MRI) and ultrasound elastography to small rodent models of disease, we aim to illustrate how in vivo imaging contributes primarily to reduction and refinement in the context of pharmacological research.

Chemokine-like receptor 1 plays a critical role in modulating the regenerative and contractile properties of muscle tissue.

Musculoskeletal diseases are a leading contributor to mobility disability worldwide. Since the majority of patients with musculoskeletal diseases present with associated muscle weakness, treatment approaches typically comprise an element of resistance training to restore physical strength. The health-promoting effects of resistance exercise are mediated via complex, multifarious mechanisms including modulation of systemic and local inflammation. Here we investigated whether targeted inhibition of the chemerin pathway, which largely controls inflammatory processes via chemokine-like receptor 1 (CMKLR1), can improve skeletal muscle function. Using genetically modified mice, we demonstrate that blockade of CMKLR1 transiently increases maximal strength during growth, but lastingly decreases strength endurance. In-depth analyses of the underlying long-term adaptations revealed microscopic alterations in the number of Pax7-positive satellite cells, as well as molecular changes in genes governing myogenesis and calcium handling. Taken together, these data provide evidence of a critical role for CMKLR1 in regulating skeletal muscle function by modulating the regenerative and contractile properties of muscle tissue. CMKLR1 antagonists are increasingly viewed as therapeutic modalities for a variety of diseases (e.g., psoriasis, metabolic disorders, and multiple sclerosis). Our findings thus have implications for the development of novel drug substances that aim at targeting the chemerin pathway for musculoskeletal or other diseases.

Enhanced tendon healing by a tough hydrogel with an adhesive side and high drug-loading capacity.

Hydrogels that provide mechanical support and sustainably release therapeutics have been used to treat tendon injuries. However, most hydrogels are insufficiently tough, release drugs in bursts, and require cell infiltration or suturing to integrate with surrounding tissue. Here we report that a hydrogel serving as a high-capacity drug depot and combining a dissipative tough matrix on one side and a chitosan adhesive surface on the other side supports tendon gliding and strong adhesion (larger than 1,000 J m-2) to tendon on opposite surfaces of the hydrogel, as we show with porcine and human tendon preparations during cyclic-friction loadings. The hydrogel is biocompatible, strongly adheres to patellar, supraspinatus and Achilles tendons of live rats, boosted healing and reduced scar formation in a rat model of Achilles-tendon rupture, and sustainably released the corticosteroid triamcinolone acetonide in a rat model of patellar tendon injury, reducing inflammation, modulating chemokine secretion, recruiting tendon stem and progenitor cells, and promoting macrophage polarization to the M2 phenotype. Hydrogels with 'Janus' surfaces and sustained-drug-release functionality could be designed for a range of biomedical applications.

Degenerative joint disease induced by repeated intra-articular injections of monosodium urate crystals in rats as investigated by translational imaging.

The objective of this work was to assess the consequences of repeated intra-articular injection of monosodium urate (MSU) crystals with inflammasome priming by lipopolysaccharide (LPS) in order to simulate recurrent bouts of gout in rats. Translational imaging was applied to simultaneously detect and quantify injury in different areas of the knee joint. MSU/LPS induced joint swelling, synovial membrane thickening, fibrosis of the infrapatellar fat pad, tidemark breaching, and cartilage invasion by inflammatory cells. A higher sensitivity to mechanical stimulus was detected in paws of limbs receiving MSU/LPS compared to saline-injected limbs. In MSU/LPS-challenged joints, magnetic resonance imaging (MRI) revealed increased synovial fluid volume in the posterior region of the joint, alterations in the infrapatellar fat pad reflecting a progressive decrease of fat volume and fibrosis formation, and a significant increase in the relaxation time T2 in femoral cartilage, consistent with a reduction of proteoglycan content. MRI also showed cyst formation in the tibia, femur remodeling, and T2 reductions in extensor muscles consistent with fibrosis development. Repeated intra-articular MSU/LPS injections in the rat knee joint induced pathology in multiple tissues and may be a useful means to investigate the relationship between urate crystal deposition and the development of degenerative joint disease.

Matrigel 3D bioprinting of contractile human skeletal muscle models recapitulating exercise and pharmacological responses.

A key to enhance the low translatability of preclinical drug discovery are in vitro human three-dimensional (3D) microphysiological systems (MPS). Here, we show a new method for automated engineering of 3D human skeletal muscle models in microplates and functional compound screening to address the lack of muscle wasting disease medication. To this end, we adapted our recently described 24-well plate 3D bioprinting platform with a printhead cooling system to allow microvalve-based drop-on-demand printing of cell-laden Matrigel containing primary human muscle precursor cells. Mini skeletal muscle models develop within a week exhibiting contractile, striated myofibers aligned between two attachment posts. As an in vitro exercise model, repeated high impact stimulation of contractions for 3 h by a custom-made electrical pulse stimulation (EPS) system for 24-well plates induced interleukin-6 myokine expression and Akt hypertrophy pathway activation. Furthermore, the known muscle stimulators caffeine and Tirasemtiv acutely increase EPS-induced contractile force of the models. This validated new human muscle MPS will benefit development of drugs against muscle wasting diseases. Moreover, our Matrigel 3D bioprinting platform will allow engineering of non-self-organizing complex human 3D MPS.

Modulation of Microglia by Voluntary Exercise or CSF1R Inhibition Prevents Age-Related Loss of Functional Motor Units.

Age-related loss of skeletal muscle innervation by motor neurons leads to impaired neuromuscular function and is a well-established clinical phenomenon. However, the underlying pathogenesis remains unclear. Studying mice, we find that the number of motor units (MUs) can be maintained by counteracting neurotoxic microglia in the aged spinal cord. We observe that marked innervation changes, detected by motor unit number estimation (MUNE), occur prior to loss of muscle function in aged mice. This coincides with gene expression changes indicative of neuronal remodeling and microglial activation in aged spinal cord. Voluntary exercise prevents loss of MUs and reverses microglia activation. Depleting microglia by CSF1R inhibition also prevents the age-related decline in MUNE and neuromuscular junction disruption, implying a causal link. Our results suggest that age-related changes in spinal cord microglia contribute to neuromuscular decline in aged mice and demonstrate that removal of aged neurotoxic microglia can prevent or reverse MU loss.

Magnetic Resonance Imaging as a Biomarker in Rodent Peripheral Nerve Injury Models Reveals an Age-Related Impairment of Nerve Regeneration.

Assessment of myelin integrity in peripheral nerve injuries and pathologies has largely been limited to post-mortem analysis owing to the difficulty in obtaining biopsies without affecting nerve function. This is further encumbered  by the small size of the tissue and its location. Therefore, the development of robust, non-invasive methods is highly attractive. In this study, we used magnetic resonance imaging (MRI) techniques, including magnetization transfer ratio (MTR), to longitudinally and non-invasively characterize both the sciatic nerve crush and lysolecithin (LCP) demyelination models of peripheral nerve injury in rodents. Electrophysiological, gene expression and histological assessments complemented the extensive MRI analyses in young and aged animals. In the nerve crush model, MTR analysis indicated a slower recovery in regions distal to the site of injury in aged animals, as well as incomplete recovery at six weeks post-crush when analyzing across the entire nerve surface. Similar regional impairments were also found in the LCP demyelination model. This research underlines the power of MTR for the study of peripheral nerve injury in small tissues such as the sciatic nerve of rodents and contributes new knowledge to the effect of aging on recovery after injury. A particular advantage of the approach is the translational potential to human neuropathies.

Tough Composite Hydrogels with High Loading and Local Release of Biological Drugs.

Hydrogels are under active development for controlled drug delivery, but their clinical translation is limited by low drug loading capacity, deficiencies in mechanical toughness and storage stability, and poor control over the drug release that often results in burst release and short release duration. This work reports a design of composite clay hydrogels, which simultaneously achieve a spectrum of mechanical, storage, and drug loading/releasing properties to address the critical needs from translational perspectives. The clay nanoparticles provide large surface areas to adsorb biological drugs, and assemble into microparticles that are physically trapped within and toughen hydrogel networks. The composite hydrogels demonstrate feasibility of storage, and extended release of large quantities of an insulin-like growth factor-1 mimetic protein (8 mg mL-1 ) over four weeks. The release rate is primarily governed by ionic exchange and can be upregulated by low pH, which is typical for injured tissues. A rodent model of Achilles tendon injury is used to demonstrate that the composite hydrogels allow for highly extended and localized release of biological drugs in vivo, while demonstrating biodegradation and biocompatibility. These attributes make the composite hydrogel a promising system for drug delivery and regenerative medicine.

Brain region-specific enhancement of remyelination and prevention of demyelination by the CSF1R kinase inhibitor BLZ945.

Multiple sclerosis (MS) is a chronic inflammatory disease affecting the central nervous system (CNS). While multiple effective immunomodulatory therapies for MS exist today, they lack the scope of promoting CNS repair, in particular remyelination. Microglia play a pivotal role in regulating myelination processes, and the colony-stimulating factor 1 (CSF-1) pathway is a key regulator for microglia differentiation and survival. Here, we investigated the effects of the CSF-1 receptor kinase inhibitor, BLZ945, on central myelination processes in the 5-week murine cuprizone model by non-invasive and longitudinal magnetic resonance imaging (MRI) and histology. Therapeutic 2-week BLZ945 treatment caused a brain region-specific enhancement of remyelination in the striatum/cortex, which was absent in the corpus callosum/external capsule. This beneficial effect correlated positively with microglia reduction, increased oligodendrocytes and astrogliosis. Prophylactic BLZ945 treatment prevented excessive demyelination in the corpus callosum by reducing microglia and increasing oligondendrocytes. In the external capsule oligodendrocytes were depleted but not microglia and a buildup of myelin debris and axonal damage was observed. A similar microglial dysfunction in the external capsule with an increase of myelin debris was obvious in triggering receptor expressed on myeloid cells 2 (TREM2) knock-out mice treated with cuprizone. Finally, therapeutic BLZ945 treatment did not change the disease course in experimental autoimmune encephalomyelitis mice, a peripherally driven neuroinflammation model. Taken together, our data suggest that a short-term therapeutic inhibition of the CSF-1 receptor pathway by BLZ945 in the murine cuprizone model enhances central remyelination by modulating neuroinflammation. Thus, microglia-modulating therapies could be considered clinically for promoting myelination in combination with standard-of-care treatments in MS patients.

Oncolytic virotherapy with an armed vaccinia virus in an orthotopic model of renal carcinoma is associated with modification of the tumor microenvironment.

Oncolytic virotherapy is an emergent promising therapeutic approach for the treatment of cancer. We have constructed a vaccinia virus (WR strain) deleted for thymidine kinase (TK) and ribonucleotide reductase (RR) genes that expressed the fusion suicide gene FCU1 derived from the yeast cytosine deaminase and uracil phosphoribosyltransferase genes. We evaluated this construct (VV-FCU1) in the orthotopic model of renal carcinoma (RenCa). Systemic administration of VV-FCU1 resulted in orthotopic tumor growth inhibition, despite temporary expression of viral proteins. VV-FCU1 treatment was associated with an infiltration of tumors by CD8+ T lymphocytes and a decrease in the proportion of infiltrating Tregs, thus modifying the ratio of CD8+/CD4+ Treg in favor of CD8+cytotoxic T cells. We demonstrated that VV-FCU1 treatment prolonged survival of animals implanted with RenCa cells in kidney. Depletion of CD8+ T cells abolished the therapeutic effect of VV-FCU1 while depletion of CD4+ T cells enhanced its protective activity. Administration of the prodrug 5-fluorocytosine (5-FC) resulted in a sustained control of tumor growth but did not extend survival. This study shows the importance of CD4+ and CD8+ T cells in vaccinia virus-mediated oncolytic virotherapy and suggests that this approach may be evaluated for the treatment of human renal cell carcinoma.

Immunotargeting of Antigen xCT Attenuates Stem-like Cell Behavior and Metastatic Progression in Breast Cancer.

Resistance to therapy and lack of curative treatments for metastatic breast cancer suggest that current therapies may be missing the subpopulation of chemoresistant and radioresistant cancer stem cells (CSC). The ultimate success of any treatment may well rest on CSC eradication, but specific anti-CSC therapies are still limited. A comparison of the transcriptional profiles of murine Her2(+) breast tumor TUBO cells and their derived CSC-enriched tumorspheres has identified xCT, the functional subunit of the cystine/glutamate antiporter system xc(-), as a surface protein that is upregulated specifically in tumorspheres. We validated this finding by cytofluorimetric analysis and immunofluorescence in TUBO-derived tumorspheres and in a panel of mouse and human triple negative breast cancer cell-derived tumorspheres. We further show that downregulation of xCT impaired tumorsphere generation and altered CSC intracellular redox balance in vitro, suggesting that xCT plays a functional role in CSC biology. DNA vaccination based immunotargeting of xCT in mice challenged with syngeneic tumorsphere-derived cells delayed established subcutaneous tumor growth and strongly impaired pulmonary metastasis formation by generating anti-xCT antibodies able to alter CSC self-renewal and redox balance. Finally, anti-xCT vaccination increased CSC chemosensitivity to doxorubicin in vivo, indicating that xCT immunotargeting may be an effective adjuvant to chemotherapy.

Revisiting fixation and embedding techniques for optimal detection of dendritic cell subsets in tissues.

Organ-specific cell types are maintained by tissue homeostasis and may vary in nature and/or frequency in pathological situations. Moreover, within a cell lineage, some sub-populations, defined by combinations of cell-surface markers, may have specific functions. Dendritic cells are the epitome of such a population as they may be subdivided into discrete sub-groups with defined functions in specific compartments of various organs. Technically, to study the distribution of DC sub-populations, it involves performing multiparametric immunofluorescence on well-conserved organ structures. However, immunodetection may be impacted by protein cross-linking and antigenic epitope masking by the use of 10% neutral-buffered formalin. To circumvent this and to preserve a good morphological tissue structure, we evaluated alternative fixatives such as Periodate Lysine Paraformaldehyde or Tris Zinc fixatives in combination with other embedding techniques. The cryosection protocols were adapted for optimal antigen detection but offered a poor morphological preservation. We therefore developed a new methodology based on Tris Zinc fixative, gelatin-sucrose embedding and freezing. Using multiple DC markers, we demonstrate that this treatment is an optimal protocol for cell-surface marker detection on high-quality tissue sections.

Lung volume quantified by MRI reflects extracellular-matrix deposition and altered pulmonary function in bleomycin models of fibrosis: effects of SOM230.

Idiopathic pulmonary fibrosis is a progressive and lethal disease, characterized by loss of lung elasticity and alveolar surface area, secondary to alveolar epithelial cell injury, reactive inflammation, proliferation of fibroblasts, and deposition of extracellular matrix. The effects of oropharyngeal aspiration of bleomycin in Sprague-Dawley rats and C57BL/6 mice, as well as of intratracheal administration of ovalbumin to actively sensitized Brown Norway rats on total lung volume as assessed noninvasively by magnetic resonance imaging (MRI) were investigated here. Lung injury and volume were quantified by using nongated or respiratory-gated MRI acquisitions [ultrashort echo time (UTE) or gradient-echo techniques]. Lung function of bleomycin-challenged rats was examined additionally using a flexiVent system. Postmortem analyses included histology of collagen and hydroxyproline assays. Bleomycin induced an increase of MRI-assessed total lung volume, lung dry and wet weights, and hydroxyproline content as well as collagen amount. In bleomycin-treated rats, gated MRI showed an increased volume of the lung in the inspiratory and expiratory phases of the respiratory cycle and a temporary decrease of tidal volume. Decreased dynamic lung compliance was found in bleomycin-challenged rats. Bleomycin-induced increase of MRI-detected lung volume was consistent with tissue deposition during fibrotic processes resulting in decreased lung elasticity, whereas influences by edema or emphysema could be excluded. In ovalbumin-challenged rats, total lung volume quantified by MRI remained unchanged. The somatostatin analog, SOM230, was shown to have therapeutic effects on established bleomycin-induced fibrosis in rats. This work suggests MRI-detected total lung volume as readout for tissue-deposition in small rodent bleomycin models of pulmonary fibrosis.

Bone- and cartilage-protective effects of a monoclonal antibody against colony-stimulating factor 1 receptor in experimental arthritis.

OBJECTIVE: Colony-stimulating factor 1 receptor (CSF-1R) essentially modulates monocyte proliferation, migration, and activation, which are considered important for the pathogenesis of rheumatoid arthritis (RA). We undertook this study to determine CSF-1R expression in human RA as well as the efficacy of a specific anti-CSF-1R monoclonal antibody (AFS98) in 2 different animal models of RA. METHODS: CSF-1R expression was examined in blood, synovium, and bone samples from RA patients, osteoarthritis (OA) patients, and healthy subjects. The efficacy of AFS98 was examined by clinical assessment, histology, and bone histomorphometry in collagen-induced arthritis (CIA) and serum-transfer arthritis. RESULTS: CSF-1R expression was increased in the synovium of RA patients compared to OA patients and healthy controls in fibroblast-like synoviocytes, follicular dendritic cells, macrophages, and osteoclasts. Circulating RA monocytes and neutrophils but not lymphocytes were CSF-1R+. In mice, blockade of CSF-1R abrogated cartilage damage, bone erosion, and systemic bone loss, and this was associated with the depletion of osteoclasts in both models. While blockade of CSF-1R did not affect inflammation in passive serum-transfer arthritis, it significantly reduced inflammation in CIA, and this was associated with the absence of synovial macrophages and reduced splenic CD11b+Gr-1- monocytes. CONCLUSION: CSF-1R was broadly expressed in human RA. Blockade of CSF-1R protected against bone and cartilage destruction in both mouse models and also showed significant antiinflammatory effects in the CIA model. These data provide evidence for CSF-1R as a therapeutic target in RA.

Therapeutic effects of anti-CD115 monoclonal antibody in mouse cancer models through dual inhibition of tumor-associated macrophages and osteoclasts.

Tumor progression is promoted by Tumor-Associated Macrophages (TAMs) and metastasis-induced bone destruction by osteoclasts. Both myeloid cell types depend on the CD115-CSF-1 pathway for their differentiation and function. We used 3 different mouse cancer models to study the effects of targeting cancer host myeloid cells with a monoclonal antibody (mAb) capable of blocking CSF-1 binding to murine CD115. In mice bearing sub-cutaneous EL4 tumors, which are CD115-negative, the anti-CD115 mAb depleted F4/80(+) CD163(+) M2-type TAMs and reduced tumor growth, resulting in prolonged survival. In the MMTV-PyMT mouse model, the spontaneous appearance of palpable mammary tumors was delayed when the anti-CD115 mAb was administered before malignant transition and tumors became palpable only after termination of the immunotherapy. When administered to mice already bearing established PyMT tumors, anti-CD115 treatment prolonged their survival and potentiated the effect of chemotherapy with Paclitaxel. As shown by immunohistochemistry, this therapeutic effect correlated with the depletion of F4/80(+)CD163(+) M2-polarized TAMs. In a breast cancer model of bone metastasis, the anti-CD115 mAb potently blocked the differentiation of osteoclasts and their bone destruction activity. This resulted in the inhibition of cancer-induced weight loss. CD115 thus represents a promising target for cancer immunotherapy, since a specific blocking antibody may not only inhibit the growth of a primary tumor through TAM depletion, but also metastasis-induced bone destruction through osteoclast inhibition.

Lymphocytic infiltration in the cutaneous lymphoma microenvironment after injection of TG1042.

BACKGROUND: Primary cutaneous lymphomas (CLs), characterized by an accumulation of clonal T or B lymphocytes preferentially localized in the skin, have been successfully treated with interferons (IFNs) which counterbalance the Th2-immunosuppressive state associated with this pathology. In a phase I/II clinical trial, we correlated the local immune infiltrate and the anti-tumor effects of repeated intralesional administrations of an adenovirus vector expressing human interferon-gamma (IFN-g) termed TG1042, in patients with advanced primary cutaneous T-cell lymphomas (CTCL) or multilesional cutaneous B-cell lymphomas (CBCL). METHODS: For each patient, variation in time of specific lymphocyte populations, defined by immunohistochemical stainings, was assessed in biopsies of injected lesions. For each patient, the change in local immune response was associated with the patient's objective response at the end of the study. RESULTS: Immunohistochemical analyses of biopsies indicate that infiltration of CD8+ T lymphocytes and of TIA-1+ cytotoxic T-cells in lesions injected with TG1042 correlates with clinical benefit. CONCLUSIONS: These data suggest for the first time that a CD8+ cytotoxic infiltrate, induced by local expression of IFN-g correlates with a clinical response. TRIAL REGISTRATION: The phase I step (TG1042.01) does not have a registration number. The phase II step (TG1042.06) registration number was NCT00394693.

In vivo preclinical low-field MRI monitoring of tumor growth following a suicide-gene therapy in an orthotopic mice model of human glioblastoma.

PURPOSE: The aim of this study was to monitor in vivo with low field MRI growth of a murine orthotopic glioma model following a suicide gene therapy. METHODS: The gene therapy consisted in the stereotactic injection in the mice brain of a modified vaccinia virus Ankara (MVA) vector encoding for a suicide gene (FCU1) that transforms a non toxic prodrug 5-fluorocytosine (5-FC) to its highly cytotoxic derivatives 5-fluorouracil (5-FU) and 5'-fluorouridine-5'monophosphate (5'-FUMP). Using a warmed-up imaging cell, sequential 3D T1 and T2 0.1T MRI brain examinations were performed on 16 Swiss female nu/nu mice bearing orthotopic human glioblastoma (U87-MG cells). The 6-week in vivo MRI follow-up consisted in a weekly measurement of the intracerebral tumor volume leading to a total of 65 examinations. Mice were divided in four groups: sham group (n=4), sham group treated with 5-FC only (n=4), sham group with injection of MVA-FCU1 vector only (n=4), therapy group administered with MVA-FCU1 vector and 5-FC (n=4). Measurements of tumor volumes were obtained after manual segmentation of T1- and T2-weighted images. RESULTS: Intra-observer and inter-observer tumor volume measurements show no significant differences. No differences were found between T1 and T2 volume tumor doubling times between the three sham groups. A significant statistical difference (p<0.05) in T1 and T2 volume tumor doubling times between the three sham groups and the animals treated with the intratumoral injection of MVA-FCU1 vector in combination with 2 weeks per os 5-FC administration was demonstrated. CONCLUSION: Preclinical low field MRI was able to monitor efficacy of suicide gene therapy in delaying the tumor growth in an in vivo mouse model of orthotopic glioblastoma.

Fusokine interleukin-2/interleukin-18, a novel potent innate and adaptive immune stimulator with decreased toxicity.

To redress the immune imbalances created by pathologies such as cancer, it would be beneficial to create novel cytokine molecules, which combine desired cytokine activities with reduced toxicities. Due to their divergent but complementary activities, it is of interest to combine interleukin-2 (IL-2) and IL-18 into one recombinant molecule for immunotherapy. Evaluation of a fusokine protein that combines murine IL-2/IL-18 shows that it is stable, maintains IL-2 and IL-18 bioactivities, has notably reduced IL-2 associated toxicities, and has a novel lymphocyte-stimulating activity. An adeno-viral expression system was used to explore the biology of this "fusokine". Inclusion of the IL-18 prosequence (proIL-18) increases the expression, secretion, and potency of this fusokine. In vivo gene transfer experiments show that Ad-IL-2/proIL-18 dramatically outdoes Ad-IL-2, Ad-proIL-18, or the combination of both, by inducing high rates of tumor rejection in several murine models. Both innate and adaptive effector mechanisms are required for this antitumor activity.

Use of adenovirus vectors for functional gene analysis in the chicken chorioallantoic membrane.

The chicken chorioallantoic membrane (CAM) assay represents one of the most widely used in vivo screening assay for genes with angiogenic (blood vessel-inducing) or angiostatic (inhibition of vessel formation or their destruction) activities. Here we show that adenovirus gene transfer vectors infect cells in the CAM and lead to expression of the viral transgene. Furthermore, infection with an adenovirus vector containing the human vascular endothelial growth factor gene induced the formation of new blood vessels. This improved method saves a considerable amount of time in the identification of genes that can influence blood vessel formation because the expensive and time-consuming production and purification of recombinant protein can be omitted.