Background Cerebral cavernous malformation (CCM) is a hemorrhagic stroke disease affecting up to 0.5% of North Americans with no approved non-surgical treatment. A subset of patients have a hereditary form of the disease due primarily to loss-of-function mutations in KRIT1, CCM2, or PDCD10. We sought to identify known drugs that could be repurposed to treat CCM. Methods and Results We developed an unbiased screening platform based on both cellular and animal models of loss-of-function of CCM2. Our discovery strategy consisted of four steps: an automated immunofluorescence and machine-learning-based primary screen of structural phenotypes in human endothelial cells deficient in CCM2; a secondary screen of functional changes in endothelial stability in these same cells; a rapid in vivo tertiary screen of dermal microvascular leak in mice lacking endothelial Ccm2; and finally a quaternary screen of CCM lesion burden in these same mice. We screened 2,100 known drugs and bioactive compounds, and identified two candidates for further study, cholecalciferol (Vitamin D3) and tempol (a scavenger of superoxide). Each drug decreased lesion burden in a mouse model of CCM vascular disease by approximately 50%. Conclusions By identifying known drugs as potential therapeutics for CCM, we have decreased the time, cost, and risk of bringing treatments to patients. Each drug also prompts additional exploration of biomarkers of CCM disease. We further suggest that the structure-function screening platform presented here may be adapted and scaled to facilitate drug discovery for diverse loss-of-function genetic vascular disease.

Cerebral cavernous malformation (CCM) is a major cerebrovascular disease affecting approximately 0.3–0.5% of the population and is characterized by enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhages. Cerebral cavernous malformation is a genetic disease that may arise sporadically or be inherited as an autosomal dominant condition with incomplete penetrance and variable expressivity. Causative loss-of-function mutations have been identified in three genes, KRIT1 (CCM1), CCM2 (MGC4607), and PDCD10 (CCM3), which occur in both sporadic and familial forms. Autophagy is a bulk degradation process that maintains intracellular homeostasis and that plays essential quality control functions within the cell. Indeed, several studies have identified the association between dysregulated autophagy and different human diseases. Here, we show that the ablation of the KRIT1 gene strongly suppresses autophagy, leading to the aberrant accumulation of the autophagy adaptor p62/SQSTM1, defective quality control systems, and increased intracellular stress. KRIT1 loss-of-function activates the mTOR-ULK1 pathway, which is a master regulator of autophagy, and treatment with mTOR inhibitors rescues some of the mole-cular and cellular phenotypes associated with CCM. Insufficient autophagy is also evident in CCM2-silenced human endothelial cells and in both cells and tissues from an endothelial-specific CCM3-knockout mouse model, as well as in human CCM lesions. Furthermore, defective autophagy is highly correlated to endothelial-to-mesenchymal transition, a crucial event that contributes to CCM progression. Taken together, our data point to a key role for defective autophagy in CCM disease pathogenesis, thus providing a novel framework for the development of new pharmacological strategies to prevent or reverse adverse clinical outcomes of CCM lesions.

The aim of this work was to study the antioxidant enzymatic defences in human follicular fluid and investigate their possible changes during reproductive ageing. To this end, we tested the specific activities and protein expression of enzymes involved in reactive oxygen species (ROS) scavenging and in detoxification of ROS byproducts in follicular fluid from young (range 27-32 years, n = 12) and older (range 39-45 years, n = 12) women participating in an IVF programme. Results show that all the tested enzymes [superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione transferase, glutathione reductase] were significantly expressed in human follicular fluid. However, when the two age groups were compared, we found that follicular fluid from older women exhibited a reduced level of glutathione transferase and catalase activities and a higher level of SOD activity. Immunoblot analysis revealed that ageing was associated with decreased protein expression of GST Pi isoform and did not affect SOD and catalase protein expression. Taken together, these findings indicate that reproductive ageing is accompanied by a change in the antioxidant enzymatic pattern that could impair ROS scavenging efficiency in the follicular environment.

KRIT1 (CCM1) is a disease gene responsible for Cerebral Cavernous Malformations (CCM), a major cerebrovascular disease of proven genetic origin affecting 0.3–0.5% of the population.Previously, we demonstrated that KRIT1 loss-of-function is associated with altered redox homeostasis and abnormal activation of the redox-sensitive transcription factor c-Jun, which collectively result in pro-oxidative, pro-inflammatory and pro-angiogenic effects, suggesting a novel pathogenic mechanism for CCM disease and raising the possibility that KRIT1 loss-of-function exerts pleiotropic effects on multiple redox-sensitive mechanisms.To address this possibility, we investigated major redox-sensitive pathways and enzymatic systems that play critical roles in fundamental cytoprotective mechanisms of adaptive responses to oxidative stress, including the master Nrf2 antioxidant defense pathway and its downstream target Glyoxalase 1 (Glo1), a pivotal stress-responsive defense enzyme involved in cellular protection against glycative and oxidative stress through the metabolism of methylglyoxal (MG). This is a potent post-translational protein modifier that may either contribute to increased oxidative molecular damage and cellular susceptibility to apoptosis, or enhance the activity of major apoptosis-protective proteins, including heat shock proteins (Hsps), promoting cell survival.Experimental outcomes showed that KRIT1 loss-of-function induces a redox-sensitive sustained upregulation of Nrf2 and Glo1, and a drop in intracellular levels of MG-modified Hsp70 and Hsp27 proteins, leading to a chronic adaptive redox homeostasis that counteracts intrinsic oxidative stress but increases susceptibility to oxidative DNA damage and apoptosis, sensitizing cells to further oxidative challenges. While supporting and extending the pleiotropic functions of KRIT1, these findings shed new light on the mechanistic relationship between KRIT1 loss-of-function and enhanced cell predisposition to oxidative damage, thus providing valuable new insights into CCM pathogenesis and novel options for the development of preventive and therapeutic strategies.

c-Src is a tyrosine kinase belonging to the Src-family kinases. It is overexpressed and/or hyperactivated in a variety of cancer cells, thus its inhibition has been predicted to have therapeutic effects in solid tumors. Recently, the pyrazolo[3,4-d]pyrimidine 3 was reported as a dual c-Src/Abl inhibitor. Herein we describe a multidisciplinary drug discovery approach for the optimization of the lead 3 against c-Src. Starting from the X-ray crystal structure of c-Src in complex with 3, Monte Carlo free energy perturbation calculations were applied to guide the design of c-Src inhibitors with improved activities. As a result, the introduction of a meta hydroxyl group on the C4 anilino ring was computed to be particularly favorable. The potency of the synthesized inhibitors was increased with respect to the starting lead 3. The best identified compounds were also found active in the inhibition of neuroblastoma cell proliferation. Furthermore, compound 29 also showed in vivo activity in xenograft model using SH-SY5Y cells.

Glioblastoma has one of the highest mortality rates among cancers, and it is the most common and malignant form of brain cancer. Among the typical features of glioblastoma tumors, there is an aberrant vascularization: all gliomas are among the most vascularized/angiogenic tumors. In recent years, it has become clear that glioblastoma cells can secrete extracellular vesicles which are spherical and membrane-enclosed particles released, in vitro or in vivo, by both normal and tumor cells; they are involved in the regulation of both physiological and pathological processes; among the latter, cancer is the most widely studied. Extracellular vesicles from tumor cells convey messages to other tumor cells, but also to normal stromal cells in order to create a microenvironment that supports cancer growth and progression and are implicated in drug resistance, escape from immunosurveillance and from apoptosis, as well as in metastasis formation; they are also involved in angiogenesis stimulation, inducing endothelial cells proliferation, and other pro-angiogenic activities. To this aim, the present paper assesses in detail the extracellular vesicles phenomenon in the human glioblastoma cell line U251 and evaluates extracellular vesicles ability to promote the processes required to achieve the formation of new blood vessels in human brain microvascular endothelial cells, highlighting that they stimulate proliferation, motility, and tube formation in a dose-response manner. Moreover, a molecular characterization shows that extracellular vesicles are fully equipped for angiogenesis stimulation in terms of proteolytic enzymes (gelatinases and plasminogen activators), pro-angiogenic growth factors (VEGF and TGFβ), and the promoting-angiogenic CXCR4 chemokine receptor.

To elucidate molecular mechanisms underlying oocyte senescence, we investigated whether oocytes from female mice of advanced reproductive age exhibit a precocious postovulatory aging that, in turn, may be responsible for the precocious activation of an apoptotic program. During a 9-h in vitro culture, the frequency of oocytes showing MII aberrations, spontaneous activation, and cellular fragmentation increased in old oocytes (P < 0.05), whereas it did not change in the young group. In old oocytes, the activities of MPF (a complex of the cyclin-dependent kinase cdc2 and cyclin B1) and MAPK (mitogen-activated protein kinase) decreased precociously, showing a first drop as early as 3 h after the beginning of in vitro culture (P < 0.05). Immunoblotting and immunocytochemical analysis revealed that, in oocytes of the old group, reduction of BCL2 expression at protein level occurred earlier than in the young group (P < 0.05) and was not associated to the loss of BCL2 transcripts detected by RT-PCR. These changes are followed by an abrupt increase of the rate of TUNEL-positive oocytes after 24 h of culture to a value of 67% +/- 6%. Exposure of young oocytes to 20 microM roscovitine or 20 microM U0126, specific inhibitors of MPF and MAPK, resulted in the decreased percentage of oocytes showing positive immunostaining for BCL2 and in an increased rate of DNA fragmentation. Present results suggest that the developmental competence of oocytes ovulated by aging mice may be negatively influenced by a downregulation of MPF and MAPK activities that in turn induces the activation of a proapoptotic signaling pathway.

BackgroundGlioblastoma recurrence after treatment with the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab is characterized by a highly infiltrative and malignant behavior that renders surgical excision and chemotherapy ineffective. It has been demonstrated that anti-VEGF/VEGFR therapies control the invasive phenotype and that relapse occurs through the increased activity of CXCR4. We therefore hypothesized that combining bevacizumab or sunitinib with the novel CXCR4 antagonist, PRX177561, would have superior antitumor activity.MethodsThe effects of bevacizumab, sunitinib, and PRX177561 were tested alone or in combination in subcutaneous xenografts of U87MG, U251, and T98G cells as well as on intracranial xenografts of luciferase tagged U87MG cells injected in CD1-nu/nu mice. Animals were randomized to receive vehicle, bevacizumab (4 mg/kg iv every 4 days), sunitinib (40 mg/kg po qd), or PRX177561 (50 mg/kg po qd).ResultsThe in vivo experiments demonstrated that bevacizumab and sunitinib increase the in vivo expression of CXCR4, SDF-1α, and TGFβ1. In addition, we demonstrate that the co-administration of the novel brain-penetrating CXCR4 antagonist, PRX177561, with bevacizumab or sunitinib inhibited tumor growth and reduced the inflammation. The combination of PRX177561 with bevacizumab resulted in a synergistic reduction of tumor growth with an increase of disease-free survival (DSF) and overall survival (OS), whereas the combination of PRX177561 with sunitinib showed a mild additive effect.ConclusionsThe CXC4 antagonist PRX177561 may be a valid therapeutic complement to anti-angiogenic therapy, particularly when used in combination with VEGF/VEGFR inhibitors. Therefore, this compound deserves to be considered for future clinical evaluation.