USE OF PI3KC2B INHIBITORS FOR THE PRESERVATION OF VASCULAR ENDOTHELIAL CELL BARRIER INTEGRITY

Abstract

Ischemic conditions are a leading cause of death for both men and women. Ischemia, a condition characterized by reduced blood flow and oxygen to an organ. Re-establishment of blood flow, or reperfusion, and re-oxygenation of the affected area following an ischemic episode is critical to limit irreversible damage. However, reperfusion also associates potentially damaging consequences. For instance, increased vascular permeability is an important contributor to edema and tissue damage following ischemic events. Here the inventors shows that genetic inhibition of PI3K-C2β reduces cerebral infarction in two ischemia/reperfusion (I/R) models and improves neurological outcome. The genetic inhibition stabilizes the blood—brain barrier (BBB) after ischemic stroke and reduces inflammation. Accordingly, the present invention relates to a method for the preservation of vascular endothelial cell barrier integrity in a patient in need thereof comprising administering to the subject a therapeutically effective amount of a PI3KC2β inhibitor.

Claims

1. A method for the preservation of vascular endothelial cell barrier integrity in a patient in need thereof comprising administering to the subject a therapeutically effective amount of a PI3KC2β inhibitor.

2. The method of claim 1 wherein the patient suffers from sepsis.

3. The method of claim 1 wherein the patient suffers from an ischemic condition.

4. The method of claim 1 wherein the patient suffers from an acute ischemic stroke.

5. The method of claim 1 wherein the PI3KC2β inhibitor is suitable for reducing infarct size, preventing or reducing edema, preventing hemorrhage and preventing no-reflow.

6. The method of claim 1 wherein the PI3KC2β inhibitor is suitable for preventing ischemia-reperfusion injuries.

7. The method of claim 1 which is performed sequentially or concomitantly with angioplasty, thrombolysis, or surgical thrombectomy.

8. The method of claim 7 wherein the thrombolysis is performed with t-PA.

9. The method of claim 1 which comprises the steps consisting of i) restoring blood supply in the ischemic tissue, and preserving the vascular endothelial cell barrier integrity of said ischemic tissue by administering to said patient a therapeutically effective amount of PI3KC2β inhibitor.

10. The method of claim 1 wherein the PI3KC2β inhibitor is a small organic molecule.

11. The method of claim 1 wherein the PI3KC2β inhibitor is an inhibitor of PI3KC2β expression.

Description

FIGURES

[0029] FIG. 1 Genetic inhibition of PI3K-C2β reduces cerebral infarction in two ischemia/reperfusion (I/R) models and improves neurological outcome. (A) Graph quantification of infarct volume measurements at 24 hours after thromboembolic stroke in wild-type (WT) and C2β.sup.D1212A/D1212A mice showing a smaller ischemic lesion in the C2β.sup.D1212A/D1212A animals (PI3KC2β KI mice) (n=17-19 mice per group; ***P<0.001, unpaired t-test). (B) Graph quantification of infarct volume measurements in wild-type (WT), heterozygous (C2β.sup.WT/D1212A) and homozygous (C2β.sup.D1212A/D1212A) mice subjected to transient middle cerebral artery occlusion (tMCAO) for 1 h followed by 24 h reperfusion (n=14-27 mice per group). **P<0.01; ***P<0.001, unpaired t-test. SHAM: operated WT mice without monofilament insertion. Results show a smaller ischemic lesion according to the level of PI3KC2β inhibition. (C) Mortality evaluation of 10-week-old C2β.sup.D1212A/D1212A and WT mice between day 0 and day 1 after tMCAO showing a better survival when PI3KC2β is inactive (n=30-39 per group). (D) Neurological scores evaluated at 24 h by Bederson (left panel) and Grip (right panel) test based on a five point system (n=27 mice per group; *P<0.05, Mann-Whitney test).

[0030] FIG. 2 Genetic inhibition of PI3K-C2β stabilizes the blood-brain barrier (BBB) after ischemic stroke and reduces inflammation. Leakage of Evans blue dye in brain parenchyma (A-B) Graph quantification of Evans blue dye extravasation measurements in wild-type (WT) and PI3KC2β KI (C2β.sup.D1212A/D1212A) mice subjected to transient middle cerebral artery occlusion (tMCAO) for 1 h followed by 24 h reperfusion (n=10-16 mice per group). **P<0.01; Mann Whitney test. (C) Genetic inhibition of PI3K-C2β reduces inflammation—Relative gene expression of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor (TNFα) 24 hours following tMCAO in the cortex and basal ganglia of control (WT) and PI3KC2(3 KI (C2β.sup.D1212A/D1212A) mice. The mRNA levels are given as the fold increase normalized to rps29 relative to the corresponding contralateral hemisphere; TNFα (n=10-14 mice per group); IL-1β, IL-6 (n=8-10 mice per group). Data represent mean ±SEM, **P<0,01; ***P=0,001; Unpaired t-test with Welch's correction. (D) Genetic inhibition of PI3K-C2β reduces neutrophils recruitment—Graph quantification for neutrophils infiltration in the ischemic hemisphere of wild-type (WT) and PI3KC2β KI (C2β.sup.D1212A/D1212A) mice subjected to tMCAO for 1 h followed by 24 h reperfusion (n=6 mice per group). **P<0.01; Mann Whitney test.(E) Representative T2-weighted MRI of WT and C2β.sup.D1212A/D1212A mice taken 24 hours after the onset of in situ clot formation by alpha thrombin, and graph quantification of area stained by P-Selectin using MPIOs in ipsilateral normalize to contralateral cortex in percentage (n=10-11 mice per group; *P<0.05; Unpaired t-test with Welch's correction).

[0031] FIG. 3 Role of endothelial versus hematopoietic PI3K-C2β in its neuroprotective effects. (A) Inhibition of endothelial PI3KC2β reduces cerebral infarction—Graph quantification of infarct volume measurements in bone marrow (BM) chimeric mice one day after tMCAO (n=13-16 mice per group) **P<0.01; ***P<0.001, unpaired t-test. WT>WT: transplantation of WT BM into WT hosts; C2β.sup.D1212A/D1212A>WT: transplantation of PI3K-C2β KI BM into WT hosts; C2β.sup.D1212A/D1212A>C2β.sup.D1212A/D1212A: transplantation of PI3K-C2β KI BM into PI3K-C2β KI hosts; WT>C2β.sup.D1212A/D1212A: transplantation of WT BM into PI3K-C2β KI hosts. A stronger protection was observed when WT BM was transplanted into PI3K-C2β KI hosts (WT>KI) suggesting that the inhibition of non hematopoietic PI3K-C2β (likely endothelial) was critical to protect from ischemic stroke lesions. (B) Inhibition of endothelial PI3KC2β reduces edema—Edema volume in chimeric mice 24 hours after tMCAO (n=13 mice per group). **P<0.01 vs WT controls; Unpaired t-test. (C) Inhibition of endothelial PI3KC2β reduces neutrophils infiltration—graph quantification for neutrophils infiltration in the ischemic hemisphere in the indicated groups 24 hours after tMCAO (WT>WT n=6; KI>WT n=4; KI>KI n=7; WT>KI n=5; Mann Whitney test).

[0032] FIG. 4 Impact of PI3K-C2β knock-down on endothelial (hCMEC/D3 cells) monolayer permeability. (A) PI3K-C2β is critical for the regulation of PI3P level in endothelial cells (1) Lysates from human brain capillary endothelial cells (hCMEC/D3) transduced with shRNA control (Sh-control) or shRNA directed against PI3K-C2β (Sh-PI3KC2β) were submitted to immunoblotting with anti-PI3KC2β antibody as indicated. Quantifications by densitometric analysis of the western blots are shown and are mean ±SEM of 6 independent experiments. (2) Graph quantification of PI3P mass assay with hCMEC control (sh-Control) or PI3K-C2β knocked-down (sh-PI3K-C2β) cells showed that PI3KC2β was responsible of PI3P production. (B) PI3K-C2β knocked-down reduces inflammation-associated endothelial permeability—Confluent endothelial hCMEC cells transduced with shRNA control (Sh-control) or shRNA directed against PI3K-C2β (Sh-PI3KC2β) were cultured on transwell and stimulated with TNFα (25 ng/ml) over the time. The transendothelial electrical resistance (TEER) was measured with a voltohmeter Millicell ERS-2. Data are shown as mean ±SEM (n=3). ***P<0.001; **P<0.01; *P<0.05, significance differences from control. (C) PI3K-C2β knocked-down maintains VE-cadherin to endothelial cell (EC) junctions in response to TNFα—Graph quantification of VE-cadherin immunoreactivities undertaken in hCMEC control (sh-Control) or knocked-down for PI3K-C2β (sh-PI3K-C2β) under basal conditions and after activation by TNFα (25 ng/ml) during 24 hours. Quantification further confirmed disrupted EC junctions in Sh-Control hCMEC cells 24 h after TNFα stimulation whereas VE-cadherin accumulated in PI3K-C2β knocked-down EC (sh-PI3K-C2β). Data represent mean ±SEM (n=5), ***P<0.001; **P<0.01; *P<0.05.

EXAMPLE

Methods

Mice

[0033] PI3K-C2β.sup.D1212A/D1212A knock-in mice and wild-type littermates bred on a C57BL/6 background were generously provided by B. Vanhaesebroeck (Alliouachene, S et al. Cell Reports 13 (9), 2015). All experiments were performed on 8- to 12-weeks-old mice, unless otherwise specified, and housed in Anexplo vivarium (US006/Regional center of functional exploration and experimental resources, Inserm/Université Paul Sabatier, Toulouse, France). Animals' procedure were approved by the institutional animal care and use committee (CEEA-122 2014-54) and conduced in accordance with the guidelines of the national institute of health.

Generation of Bone Marrow Chimeric Mice

[0034] The recipient mice were irradiated to the non-invasive exploration platform located at the Nuclear Medicine Department of the Rangueil Hospital (Biobeam Biological Irradiator 8000). The animals received a single dose of 9 Gray (Gy) for 6 min and their immune system rescued by bone marrow transplantation from either WT or PI3K-C2β KI donors after 24 h in ventilated cages with drinking water supplemented with 10% antibiotics Baytril (Bayer). The tMCAO surgery was performed approximately 4 weeks later.

tMCAO versus Thromboembolic Stroke Mice Model

[0035] To investigate the functional role of class II PI3K-C2β in reperfusion injury induced by ischemic stroke we use the mechanical mouse model of tMCAO and the model of thromboembolic stroke. These two models provide powerful experimental approaches for translational stroke research and are representative of two different clinical situations. The first model results in prompt recirculation, mimicking cerebrovascular surgery or interventional thrombectomy, whereas the second mimics the cellular and molecular mechanisms of thrombosis and thrombolysis with tissue-type plasminogen activator (rt-PA), resulting in the gradual restoration of the recirculation. These two models provide powerful experimental approaches for translational stroke research and are representative of the two different clinical situations.

Transient Middle Cerebral Artery Occlusion (tMCAO)

[0036] Mice were anesthetized with 3% isoflurane in a mixture of 70% N2O/30% O2 for cerebral focal ischemia-reperfusion induction by tMCAO according to the established procedure Braeuninger et al., Methods Mol Biol. 2012;788:29-42). After midline neck incision, the internal carotid artery was occluded with an 18-mm length of 4-0 nylon monofilament with a flame-rounded tip to occlude the origin of the Middle Cerebral Artery (MCA). After 1 h occlusion, mice were reanesthetized, the suture and ligatures were removed to initiate reperfusion for 24 h. Successful induction of focal ischemia was confirmed by contralateral hemiparesis. Exclusion criteria were excessive bleeding or death within 24 h after tMCAO.

Thromboembolic Stroke

[0037] Mice were anesthetized with isoflurane (4-5% for induction, 1-2% thereafter) in a 70% N2O/30% O2 gas mixture. Thereafter, they are placed in a stereotaxic frame, the skin between the right eye and the right ear is incised, and the temporal muscle is retracted. A small craniotomy is performed, the dura is excised, and the middle cerebral artery (MCA) exposed. The pipette (glass micro-pipette, tip size 30-50 μm) is introduced into the lumen of the artery and 1 μL of murine α-thrombin (Haematologic Technologies Inc., Stago BNL, NL) is injected to induce in situ clot formation (Orset C, Stroke. 2007;38(10):2771-2778). The pipette is not removed for 10 min after the injection of thrombin to allow the clot stabilization. The rectal temperature is maintained at 37±0.5° C. throughout the surgical procedure using a feedback-regulated heating system. Cerebral blood flow velocity (CBFv) is used as an occlusion index (blood flow is reduced by up to 60% of baseline) and is monitored using a laser Doppler within the MCA territory on the dorsal face of the skull over 60 min. These experiments were performed in the Experimental Stroke Research Platform (ESRP, Caen, France).

In Vivo Brain Imaging

[0038] In vivo brain imaging is performed in the Biomedical Imaging Platform (Cyceron, Caen, France) using a 7t MRI (Brucker, pharmascan) on anesthetized mice (2% isoflurane in a 70% nitrous oxide and 30% oxygen mixture), 24 h post-occlusion. For this purpose, a set of sequences in the axial plan including time-of-flight angiography, T2-weighted (T2W), and T2*-weighted (T2*W) imaging will be performed. These sequences allow the assessment of arterial recanalization, ischemic infarction, and brain hemorrhages, respectively. Images are then post-processed using imageJ software for ischemic calculation and angiographic score measurements.

Evans Blue Extravasation

[0039] The integrity of the blood brain barrier (BBB) was assessed by measuring extravasation of Evans blue dye into the brain parenchyma. A 2% solution of Evans blue in saline was injected intravenously at 4 mL/kg 1 h after induction of tMCAO. Twenty four hours later, mice were anesthetized with isoflurane and perfused with saline through the left cardiac ventricle until infusion fluid was colorless. Mice were sacrificed, brains were removed and 2-mm coronal sections were sliced for photography.

Immunohistochemistry

[0040] Front and rear portions of each brain that were postfixed for 48 hours at 4° C. in 10% neutral buffered formalin (Sigma), embedded in paraffin, and sectioned at a thickness of 10 μm. Tissue sections were mounted on pretreated slides and deparaffinized in xylene. Hematoxylin-and-eosin (HE) staining was performed on selected sections from each brain to assess the degree of leukocyte infiltration.

RNA Extraction and Reverse Transcription

[0041] Tissues were homogenized and total RNA were extracted in Trizol reagent (Life Technologies, Gaithersburg, MD, U.S.A.) according to the manufacturer's suggested protocol. Total RNA concentration was determined from spectrophotometric optical density measurement (260 and 280 nm). Reverse transcriptase reactions were then carried out using the RNA PCR Core Kit (GeneAmp RNA PCR Core kit, ThermoFischer Scientific). Experiments were realized according to the manufacturer's suggested protocol and were carried out in a DNA Thermal Cycler 480 (Perkin Elmer, Branchburg, NJ, U.S.A.). The cDNA was then stored at −20° C.

[0042] The cDNA sequences for RPS29 (ribosomal protein small subunit 29), interleukin −1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) were obtained from GeneBank. The primer and probe sequences used are reported in Table 1. Real-time PCR was performed using the TaqMan Universal PCR Master Mix. All samples were run in duplicate and the output level reported as the average of the two duplicate. Amplification conditions were performed using ABI PRISM 7700 sequence detection system (PE Applied Biosystems). The threshold cycle, which represents the PCR cycle at which an increase in reporter fluorescence above background is first detected, was determined by the software, based on the standard curves.

[0043] Using the formula provided by the manufacturer (PE Applied Biosystems) and described by Wang et al. (Wang et al. Journal of Neuroscience Research, 2000; 59: 238-246, Wang et al. J Cereb Blood Flow Metab, 2000; 20: 15-20), the values were extrapolated to calculate the relative number of mRNA copies as compared with RPS29 levels as control. The data are presented as the mean ±SD. ANOVA followed by Tukey post hoc analysis was used to evaluate differences between time points. Student's t-tests were used to evaluate differences between left and right hemispheres.

Cell Culture

[0044] Immortalized human brain capillary endothelial cells (hCMEC/D3 cell line), which retain the characteristics of the cerebral circulation (Weksler, B. B. et al. The FASEB Journal, 2005,19, n° 13: 1872-74.), were cultured in rat tail collagen I (Cultrex, Trevigen, France) coated plates (1.5 mg/mL) in medium consisting of EndoGRO medium (Merck Millipore) supplemented with a dedicated supplement (EndoGRO MV Supplement Kit, Merck Millipore), 1 ng/mL basal Fibroblast Growth Factor (Sigma-Aldrich) and 1% penicillin—Streptomycin (Invitrogen). Cells were cultured in an incubator at 37° C. with 5% CO.sub.2 and saturated humidity. From these cells, a batch having integrated a vector by lentiviral transduction was created. HCMEC/D3 pLKO-ShRNA PI3KC2β (shRNA-PI3K-C2β) cells having integrated a shRNA directed against PI3K-C2β. For the cells transduced by the shRNA-PI3K-C2β or shRNA-control lentiviruses the medium was supplemented with 3 μg/mL puromycin. The cells were passed twice a week with Trypsin/EDTA (Sigma-Aldrich) or accutase (BD Pharmingen). The cells were counted using a cell counter (Z1 coulter particle counter Beckman Coulter Brea USA).

Trans-Endothelial Electrical Resistance (TEER)

[0045] For trans-endothelial electrical resistance hCMEC/D3 were seeded on type I collagen pre-coated Transwell-Clear filters (Costar, Corning Incorporation). Assay medium was changed after 4 and 7 days and transport assays were performed when cells form monolayers (7-10 days after seeding). Culture systems on inserts were exposed to treatment (hrTNFα at 25 ng/ml), and TEER were measured using an epithelial volt-ohmmeter (Millicell). The resistance of ECM-coated inserts was used as control. The values obtained were plotted on GraphPad software and checked for significance.

Western Blot Analysis

[0046] Proteins were extracted from tissues in lysis buffer containing 150 mM NaCl, 20 mM Tris.HCl pH7.4, 1% Triton X-100, 0.2% SDS, 4 mM EDTA, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mM Na3VO4, 1 mM PMSF. The homogenate was cleared by centrifugation at 4□C for 20 min at 13,000 g and the supernatant fraction recovered. Protein concentration was determined by colorimetric assay (BCA, Pierce). Homogenates were resolved by SDS-PAGE, transferred to nitrocellulose membranes and probed with antibodies to PI3K-C2β (1:1000) from BD Biosciences (#611342) overnight at 4° C. Antigen-specific binding of antibodies was visualized by ECL.

Immunofluorescence

[0047] Cells were seeded at 2.5×104 cells.cm.sup.−2 in collagen I-coated glass coverslip in 24 well plates. After snap wash in PBS, cells were fixed in 4% formaldehyde and permeabilized with 0.1% Triton X-100. Cells were blocked in PBS with 1% BSA fatty acid-free 1 h and incubated with VE-Cadherin primary antibody (#555661, BD Pharmingen) in blocking solution 2 h at RT in humid chamber. After washes, lamellae are incubated with the appropriate fluorescent secondary antibody and DAPI to evaluate cell number. Coverslips were mounted on glass slides with Mowiol mounting solution. Confocal images were captured with a LSM780 operated with Zen software (Carl Zeiss). Profiling of fluorescence intensity was carried out with ImageJ (National Institute of Health, Bethesda, MA, USA).

Mass Assay

[0048] PI3P levels were quantified by a mass assay as previously described (Chicanne, G. et al. Biochemical Journal. 2012, 447, n° 1: 17-23). Preparation of cell extract for mass assay was as follows. After removing media, cells were immediately scraped off and recovered in ice-cold 1M HCl, followed by centrifugation at 2000 rpm at 4° C. and snap-freezing of the cell pellet. Samples were stored at −80° C. before processing for PI3P mass assay.

Statistical Analysis

[0049] All data are shown as mean +/−S.E.M. The statistical significance of differences between means was calculated by one-way anova, two-way anova or t-test analysis, as appropriate. Statistical significance was assumed at p<0.05 and indicated as *p<0.05, **p<0.01, ***p<0.001 realize using Prism Software (GraphPad, version 5).

Results

[0050] The results are depicted in FIGS. 1-4.

[0051] Firstly, the results show that genetic inhibition of PI3K-C2β reduces the cerebral infarction in two ischemia/reperfusion (UR) models and improves neurological outcome. C2β.sup.D1212A/D1212A mice displayed a significantly improved outcome compared to WT mice resulting in a significant increase in survival, a better overall neurologic function 24 hours after tMCAO (Bederson score: mean, 2.82 for WT vs 2.04 for C2β.sup.D1212A/D1212A; P<0.05) and an improved motor function and coordination (grip test score: mean, 2.26 for wild-type vs 3.07 for C2β.sup.D1212A/D1212A; P<0.05) (FIG. 1). Collectively, these data demonstrate that the marked reduction of infarct volume in C2β.sup.D1212A/D1212A mice was functionally relevant.

[0052] Genetic inhibition of PI3K-C2β stabilizes the blood—brain barrier (BBB after ischemic stroke and reduces inflammation. In the thromboembolic stroke model, ultrasensitive molecular MRI of cerebrovascular inflammatory molecules expressed by endothelial cells, such as adhesion molecule P-selectin, was used to evaluate the degree of brain inflammation in vivo. Antibody-based microsized particles of iron oxide (MPIOs) targeting P-Selectin were injected intravenously in mice 24 h after induction of acute thrombosis in the MCA. MRI was acquired 20 min after intravenous administration of targeted MPIOs. Absence of PI3KC2β activity (C2β.sup.D1212A/D1212A mice) efficiently protected from endothelial P-Selectin expression compared to WT mice (2.26% vs 5.18%) indicating a decrease of endovascular inflammation (FIG. 2). The results also show that inhibition of endothelial PI3KC2β reduces cerebral infarction, edema and neutrophils infiltration (FIG. 3). In human cerebral microvascular endothelial hCMEC/D3 cells, the results show that PI3K-C2β is critical for the regulation of PI3P level and PI3K-C2β knocked-down reduces inflammation associated endothelial permeability. PI3K-C2β knocked-down maintains VE-cadherin to endothelial cell (EC) junctions in response to TNFα (FIG. 4)

[0053] Altogether these results highlight the involvement of PI3K-C2β in infarct generation and CNS inflammation in two different models of stroke and demonstrate that inhibition of this lipid kinase is beneficial in acute ischemic stroke.

REFERENCES

[0054] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.