PARIN5, a Novel Thrombin Receptor Antagonist Modulates a Streptozotocin Mice Model for Diabetic Encephalopathy.

Diabetic encephalopathy (DE) is an inflammation-associated diabetes mellitus (DM) complication. Inflammation and coagulation are linked and are both potentially modulated by inhibiting the thrombin cellular protease-activated receptor 1 (PAR1). Our aim was to study whether coagulation pathway modulation affects DE. Diabetic C57BL/6 mice were treated with PARIN5, a novel PAR1 modulator. Behavioral changes in the open field and novel object recognition tests, serum neurofilament (NfL) levels and thrombin activity in central and peripheral nervous system tissue (CNS and PNS, respectively), brain mRNA expression of tumor necrosis factor α (TNF-α), Factor X (FX), prothrombin, and PAR1 were assessed. Subtle behavioral changes were detected in diabetic mice. These were accompanied by an increase in serum NfL, an increase in central and peripheral neural tissue thrombin activity, and TNF-α, FX, and prothrombin brain intrinsic mRNA expression. Systemic treatment with PARIN5 prevented the appearance of behavioral changes, normalized serum NfL and prevented the increase in peripheral but not central thrombin activity. PARIN5 treatment prevented the elevation of both TNF-α and FX but significantly elevated prothrombin expression. PARIN5 treatment prevents behavioral and neural damage in the DE model, suggesting it for future clinical research.

LPS-Induced Coagulation and Neuronal Damage in a Mice Model Is Attenuated by Enoxaparin.

Background. Due to the interactions between neuroinflammation and coagulation, the neural effects of lipopolysaccharide (LPS)-induced inflammation (1 mg/kg, intraperitoneal (IP), n = 20) and treatment with the anti-thrombotic enoxaparin (1 mg/kg, IP, 15 min, and 12 h following LPS, n = 20) were studied in C57BL/6J mice. Methods. One week after LPS injection, sensory, motor, and cognitive functions were assessed by a hot plate, rotarod, open field test (OFT), and Y-maze. Thrombin activity was measured with a fluorometric assay; hippocampal mRNA expression of coagulation and inflammation factors were measured by real-time-PCR; and serum neurofilament-light-chain (NfL), and tumor necrosis factor-α (TNF-α) were measured by a single-molecule array (Simoa) assay. Results. Reduced crossing center frequency was observed in both LPS groups in the OFT (p = 0.02), along with a minor motor deficit between controls and LPS indicated by the rotarod (p = 0.057). Increased hippocampal thrombin activity (p = 0.038) and protease-activated receptor 1 (PAR1) mRNA (p = 0.01) were measured in LPS compared to controls, but not in enoxaparin LPS-treated mice (p = 0.4, p = 0.9, respectively). Serum NfL and TNF-α levels were elevated in LPS mice (p < 0.05) and normalized by enoxaparin treatment. Conclusions. These results indicate that inflammation, coagulation, neuronal damage, and behavior are linked and may regulate each other, suggesting another pharmacological mechanism for intervention in neuroinflammation.

Intrinsic Expression of Coagulation Factors and Protease Activated Receptor 1 (PAR1) in Photoreceptors and Inner Retinal Layers.

The aim of this study was to characterize the distribution of the thrombin receptor, protease activated receptor 1 (PAR1), in the neuroretina. Neuroretina samples of wild-type C57BL/6J and PAR1-/- mice were processed for indirect immunofluorescence and Western blot analysis. Reverse transcription quantitative real-time PCR (RT-qPCR) was used to determine mRNA expression of coagulation Factor X (FX), prothrombin (PT), and PAR1 in the isolated neuroretina. Thrombin activity following KCl depolarization was assessed in mouse neuroretinas ex vivo. PAR1 staining was observed in the retinal ganglion cells, inner nuclear layer cells, and photoreceptors in mouse retinal cross sections by indirect immunofluorescence. PAR1 co-localized with rhodopsin in rod outer segments but was not expressed in cone outer segments. Western blot analysis confirmed PAR1 expression in the neuroretina. Factor X, prothrombin, and PAR1 mRNA expression was detected in isolated neuroretinas. Thrombin activity was elevated by nearly four-fold in mouse neuroretinas following KCl depolarization (0.012 vs. 0.044 mu/mL, p = 0.0497). The intrinsic expression of coagulation factors in the isolated neuroretina together with a functional increase in thrombin activity following KCl depolarization may suggest a role for the PAR1/thrombin pathway in retinal function.

Suprachoroidal delivery of bevacizumab in rabbit in vivo eyes: Rapid distribution throughout the posterior segment.

PURPOSE: To test the in-vivo bio-distribution and safety of bevacizumab delivery into the suprachoroidal space (SCS) using a novel injection system in a large eye model. METHODS: Bevacizumab (1.25 mg) was injected into the vitreous (IVT, 50 µL, n = 12) or the SCS, (150 µL, n = 37) of live rabbits. Immunofluorescence and ELISA were used to assess bevacizumab distribution. Intraocular pressure (IOP) measurements, SD-OCT and fundus imaging, electroretinogram, and histology analysis were performed for safety assessment. RESULTS: Bevacizumab was observed throughout the choroid layers up to the retinal pigment epithelium (RPE), within 1 h following SCS injection. The Cmax of bevacizumab in the retina/choroid was 1043 ± 597 µg/gr tissue (mean ± standard error), 40-fold higher than in IVT injected eyes (p = 0.0339). One day following SCS injection, bevacizumab was detected throughout the posterior pole with a two-fold lower concentration. One week post-SCS injection, bevacizumab concentration in the retina/choroid dropped to 2.36 ± 1.32 µg/gr tissue (p = 0.034 vs. 1 h), with a half-life of 20 h. No suprachoroidal blebs, retinal detachment, hemorrhages, inflammation or changes in retinal function were observed up to 2 months following SCS injection. Elevated IOP (+16 mmHg) was observed two minutes post-SCS injection and spontaneously returned to baseline levels within 10 minutes. CONCLUSIONS: The novel injection system enabled a minimally invasive, safe, and consistent delivery of bevacizumab with rapid distribution throughout the choroid layers up to the RPE in large eyes. Large volumes of anti-angiogenic are delivered in close proximity to the retina due to the high volume distribution.

Efficacy and safety of injecting increasing volumes into the extravascular spaces of the choroid using a blunt adjustable depth injector.

PURPOSE: To evaluate the efficacy and safety of injecting increasing volumes into the extravascular spaces of the choroid (EVSC) in rabbit eyes in vivo using a blunt adjustable depth injector. METHODS: Indocyanine green (ICG) was injected in the superior-temporal quadrant, 2 mm posterior to the limbus at increasing volumes (0.1-0.3 ml) into the EVSC of New Zealand rabbit eyes in vivo. Intraocular pressure (IOP) measurements, spectral domain optical coherence tomography (SD-OCT), fundus imaging and histology analysis were performed to assess the safety and efficacy of the injection. RESULTS: Volumes up to 0.3 ml were administered consistently. ICG injection was successfully monitored in vivo using infrared fundus imaging and SD-OCT. ICG was detected across the EVSC compartment, reaching the retinal pigment epithelium, optic nerve head and visual streak. Injection of 0.3 ml yielded maximal dye distribution with a coverage area of 61.8% ± 6.7% (mean ± standard error, SE) of the posterior segment. Maximal IOP elevation was recorded 5 min following injection of 0.2 and 0.3 ml ICG (+ 20.0 mmHg, + 19.4 mmHg, respectively). Twenty minutes post-injection, the IOP was < 15 mmHg in all injection volumes. No retinal detachment or hemorrhages were detected in any of the injected eyes. CONCLUSIONS: This study demonstrates consistent and safe delivery of large volumes within the EVSC using a blunt adjustable depth injector that distributes the dye over 60% of the retinal surface. This injection system may offer a minimally invasive and easy way to deliver large volumes of pharmaceuticals into the posterior segment.

Repetitive magnetic stimulation protects corneal epithelium in a rabbit model of short-term exposure keratopathy.

PURPOSE: To investigate the effect of repetitive magnetic stimulation (RMS) on corneal epithelial permeability in a rabbit model of exposure keratopathy. METHODS: 61 female New Zealand White (NZW) rabbits were treated on one eye with repetitive magnetic stimulation (RMS) at a frequency of 20 Hz for 15 min. The other eye was untreated. Rabbit eyes were kept open for 2 h to induce acute corneal desiccation. The extent of fluorescein corneal staining was evaluated using EpiView software and the concentration of fluorescein in the anterior chamber was determined by a fluorometer. Safety was evaluated by electroretinogram, spectral domain optical coherence tomography (SD-OCT) and histopathology. Expression pattern of corneal cell markers was determined by immunofluorescence. RESULTS: A significant decrease in fluorescein concentration in the anterior chamber (54 ±â€¯8.4 ng/ml vs. 146.5 ±â€¯18.6 ng/ml, p = 0.000001) and in corneal surface fluorescein staining score (1.7 ±â€¯0.2 vs. 4.6 ±â€¯0.6, p = 0.00001) was obtained in RMS-treated eyes compared with control eyes, respectively. RMS treatment reduced by nearly 4 fold the percentage of corneal area with epithelial erosions by anterior segment SD-OCT. The therapeutic effect was maintained for at least 3 months. Increased expression of epithelial tight junction protein Zo-1 was observed in treated eyes. SD-OCT and histopathology analysis revealed no pathological changes in the treated or non-treated eyes. CONCLUSIONS: RMS treatment decreases epithelial corneal erosions in a rabbit model of exposure keratopathy, with no indication of pathological changes. RMS may present a novel treatment for protection of corneal epithelium from desiccation.

The p53-Mdm2 loop: a critical juncture of stress response.

The presence of a functional p53 protein is a key factor for the proper suppression of cancer development. A loss of p53 activity, by mutations or inhibition, is often associated with human malignancies. The p53 protein integrates various stress signals into a growth restrictive cellular response. In this way, p53 eliminates cells with a potential to become cancerous. Being a powerful decision maker, it is imperative that p53 will be activated properly, efficiently and temporarily in response to stress. Equally important is that p53 activation will be extinguished upon recovery from stress, and that improper activation of p53 will be avoided. Failure to achieve these aims is likely to have catastrophic consequences for the organism. The machinery that governs this tight regulation is largely based on the major inhibitor of p53, Mdm2, which both blocks p53 activities and promotes its destabilization. The interplay between p53 and Mdm2 involves a complex network of positive and negative feedback loops. Relief from Mdm2 suppression is required for p53 to be stabilized and activated in response to stress. Protection from Mdm2 entails a concerted action of modifying enzymes and partner proteins. The association of p53 with the PML-nuclear bodies may provide an infrastructure in which this complex regulatory network can be orchestrated. In this chapter we use examples to illustrate the regulatory machinery that drives this network.

T cell survival and function requires the c-Abl tyrosine kinase.

C-Abl (Abl) regulates multiple cellular processes, including proliferation, survival, shape determination and motility, and participates in cellular responses to genotoxic and oxidative stress stimuli. Mice lacking Abl exhibit retarded growth, osteoporosis and defects in the immune system resulting in lymphopoenia and susceptibility to infections, leading to early death. To define the role of Abl in the regulation of adult T cells we ablated Abl exclusively in T cells by generating mice with floxed abl alleles and expressing an Lck-Cre transgene (Abl-T(-/-)). These mice exhibited thymic atrophy and abnormally reduced T cell numbers in the periphery. The thymic atrophy was caused by increased susceptibility of thymocytes to cell death. Importantly, Abl deficient T cells displayed abnormally reduced response to mitogenic stimulation in vitro. Consequently, Abl-T(-/-) mice exhibited impaired ability to reject syngeneic tumor, to induce T-mediated tumor cell killing, and to generate anti-tumor antibodies. These results demonstrate a cell-autonomous role for Abl in T cell function and survival.

C-Abl as a modulator of p53.

P53 is renowned as a cellular tumor suppressor poised to instigate remedial responses to various stress insults that threaten DNA integrity. P53 levels and activities are kept under tight regulation involving a complex network of activators and inhibitors, which determine the type and extent of p53 growth inhibitory signaling. Within this complexity, the p53-Mdm2 negative auto-regulatory loop serves as a major route through which intra- and extra-cellular stress signals are channeled to appropriate p53 responses. Mdm2 inhibits p53 transcriptional activities and through its E3 ligase activity promotes p53 proteasomal degradation either within the nucleus or following nuclear export. Upon exposure to stress signals these actions of Mdm2 have to be moderated, or even interrupted, in order to allow sufficient p53 to accumulate in an active form. Multiple mechanisms involving a variety of factors have been demonstrated to mediate this interruption. C-Abl is a critical factor that under physiological conditions is required for the maximal and efficient accumulation of active p53 in response to DNA damage. C-Abl protects p53 by antagonizing the inhibitory effect of Mdm2, an action that requires a direct interplay between c-Abl and Mdm2. In addition, c-Abl protects p53 from other inhibitors of p53, such as the HPV-E6/E6AP complex, that inhibits and degrades p53 in HPV-infected cells. Surprisingly, the oncogenic form of c-Abl, the Bcr-Abl fusion protein in CML cells, also promotes the accumulation of wt p53. However, in contrast to the activation of p53 by c-Abl, its oncogenic form, Bcr-Abl, counteracts the growth inhibitory activities of p53 by modulating the p53-Mdm2 loop. Thus, it appears that by modulating the p53-Mdm2 loop, c-Abl and its oncogenic forms critically determine the type and extent of the cellular response to DNA damage.

Treatment of chronic myeloid leukemia cells with imatinib (STI571) impairs p53 accumulation in response to DNA damage.

Chronic myelogenous leukaemia (CML) is induced by the Bcr-Abl fusion protein. Inhibition of Bcr-Abl by STI571 is widely used to treat CML patients. Unlike in most cancer types, the frequency of p53 mutations in CML is low. Here, we investigated the effect of STI571 treatment of CML cells on p53 regulation. Exposure of CML cells, including established cell lines and freshly isolated cells from patients, to STI571 reduced p53 protein levels, and severely impaired its accumulation in response to DNA damage. This may be explained by the status of p53 serine 20 phosphorylation. In non-stressed CML cells, serine 20 of p53 is constitutively phosphorylated by Chk1, and is inhibited by STI571. In response to DNA damage, however, this phosphorylation is mediated by Chk1 and Chk2, and is only partially inhibited by STI571. CML cells expressing wild-type p53 are more resistant to treatment with STI571, but moderately more sensitive to DNA damage, than CML cells lacking p53. An enhanced induction of apoptosis by STI571 and DNA damage is observed in CML cells bearing wild-type p53, but not in cells lacking functional p53. This implies that the status of p53 may affect the response of CML cells to this combined treatment.

Apoptosis - the p53 network.

Exposure to cellular stress can trigger the p53 tumor suppressor, a sequence-specific transcription factor, to induce cell growth arrest or apoptosis. The choice between these cellular responses is influenced by many factors, including the type of cell and stress, and the action of p53 co-activators. p53 stimulates a wide network of signals that act through two major apoptotic pathways. The extrinsic, death receptor pathway triggers the activation of a caspase cascade, and the intrinsic, mitochondrial pathway shifts the balance in the Bcl-2 family towards the pro-apoptotic members, promoting the formation of the apoptosome, and consequently caspase-mediated apoptosis. The impact of these two apoptotic pathways may be enhanced when they converge through Bid, which is a p53 target. The majority of these apoptotic effects are mediated through the induction of specific apoptotic target genes. However, p53 can also promote apoptosis by a transcription-independent mechanism under certain conditions. Thus, a multitude of mechanisms are employed by p53 to ensure efficient induction of apoptosis in a stage-, tissue- and stress-signal-specific manner. Manipulation of the apoptotic functions of p53 constitutes an attractive target for cancer therapy.

Tyrosine phosphorylation of Mdm2 by c-Abl: implications for p53 regulation.

The p53 tumor suppressor is inhibited and destabilized by Mdm2. However, under stress conditions, this downregulation is relieved, allowing the accumulation of biologically active p53. Recently we showed that c-Abl is important for p53 activation under stress conditions. In response to DNA damage, c-Abl protects p53 by neutralizing the inhibitory effects of Mdm2. In this study we ask whether this neutralization involves a direct interplay between c-Abl and Mdm2, and what is the contribution of the c-Abl kinase activity? We demonstrate that the kinase activity of c-Abl is required for maintaining the basal levels of p53 expression and for achieving maximal accumulation of p53 in response to DNA damage. Importantly, c-Abl binds and phosphorylates Mdm2 in vivo and in vitro. We characterize Hdm2 (human Mdm2) phosphorylation at Tyr394. Substitution of Tyr394 by Phe394 enhances the ability of Mdm2 to promote p53 degradation and to inhibit its transcriptional and apoptotic activities. Our results suggest that phosphorylation of Mdm2 by c-Abl impairs the inhibition of p53 by Mdm2, hence defining a novel mechanism by which c-Abl activates p53.