TRPM4-specific blocking antibody attenuates reperfusion injury in a rat model of stroke
Reperfusion therapy is currently the gold standard treatment for acute ischemic stroke. However, reperfusion injuries such as oedema and haemorrhagic transformation largely limit the use of this potent treatment to a narrow time window. Recently, transient receptor potential melastatin 4 (TRPM4) channel has emerged as a potential target for vascular protection in stroke management. Non-specificity and side effects are major concerns for current TRPM4 blockers. The present study was undertaken to develop a novel TRPM4 blocker for stroke management. We report the generation of a TRPM4-specific antibody M4P which binds to a region close to the channel pore. M4P could inhibit TRPM4 current and downregulate TRPM4 surface expression, therefore prevent hypoxia-induced cell swelling. In the rat model of 3-h stroke reperfusion, application of M4P at 2 h after occlusion ameliorated reperfusion injury by improving blood–brain barrier integrity, and enhanced functional recovery. Our results demonstrate that TRPM4 blockade could attenuate reperfusion injury in stroke recanalization. When applied together with reperfusion treatments, TRPM4 blocking antibody has the potential to extend the therapeutic time window for acute ischemic stroke.
KeywordsIschemic stroke TRPM4 Blood–brain barrier Reperfusion injury Time window
Transient receptor potential melastatin 4 (TRPM4) is a voltage-dependent, nonselective monovalent cation channel which is activated by elevated cytosolic Ca2+, and modulated by ATP depletion . Under hypoxic conditions, its expression is upregulated, and channel activities are enhanced by oxygen depletion, leading to oncotic cell death . Therefore, TRPM4 inhibition could alleviate oedema formation by stabilising the blood–brain barrier (BBB) . Recently, TRPM4 has emerged as a therapeutic target for many brain disorders such as stroke, spinal cord injury, and head injury.
TRPM4 has been reported to interact with sulfonylurea receptor-1 (Sur1) to form a SUR1-TRPM4 channel complex, and application of SUR1 blockers sulphonylureas could inhibit SUR1-TRPM4 function . SUR1 is an auxiliary subunit of KATP channel which senses ATP changes in pancreatic β cells, and regulates insulin secretion . Therefore, sulphonylureas such as glibenclamide are widely used to control blood glucose level in diabetic patients. As sulphonylureas are being used in clinical practice, multiple studies on stroke patients with or without diabetes mellitus were carried out using glibenclamide. The result showed that use of sulphonylureas before or after stroke onset could reduce haemorrhage transformation, attenuate cerebral oedema, and improve neurological outcome . In contrast, other studies on diabetic patients who later developed stroke revealed that application of sulphonylureas achieved no better outcome than other anti-diabetic treatments [7, 8]. Such controversies may arise from differences in patient inclusion criteria, dose of sulphonylureas, or the severity of diabetes mellitus. Interestingly, a study showed that the application of sulfonylurea glimepiride achieved neuroprotection against stroke only in normal mice but not in type 2 diabetic mice , suggesting that the presence of diabetes mellitus could be a confounding factor for the use of sulphonylureas to manage stroke. Recently, a phase II multicentre clinical trial using glibenclamide in patients with large anterior circulation hemispheric infarction was reported , and there was no difference between the glibenclamide and control groups for the primary outcome, even though signs of oedema alleviation were observed. One possible reason is that the dose of glibenclamide used was low in the study, as a high dose could induce persistent hypoglycaemia in patients .
In view of these issues, we seek to block TRPM4 directly without targeting SUR1. The expression of TRPM4 can be inhibited at the mRNA level with gene-specific siRNA. TRPM4 siRNA could enhance vascular integrity and improve motor functions in both permanent and transient middle cerebral artery occlusion (MCAO) models [12, 13]. As siRNA functions at the mRNA level, the time of application is critical. Once TRPM4 protein is upregulated, the therapeutic effect of siRNA becomes less effective. In this study, we describe the production of a TRPM4-specific blocking antibody M4P and demonstrate that M4P could improve stroke outcome in stroke reperfusion model. As M4P does not interact with SUR1/KATP channel complex, potential side effects from glibenclamide can be avoided.
Material and methods
Generation of polyclonal antibody M4P
The procedure to generate rabbit polyclonal antibody M4P is similar to our previous report [13, 14]. In brief, DNA sequence encoding rat TRPM4 polypeptide antigen (Fig. 1a in the online-only Data Supplement) was cloned in frame into pGEX-4T-1 plasmid. GST-fused protein was purified with glutathione-agarose (G4510, Sigma-Aldrich, MI, USA) (Fig. 1c in the online-only Data Supplement). 0.5 mg purified protein was injected into female New Zealand White rabbits subcutaneously once a month. Complete Freund’s adjuvant was used for the first immunisation, and incomplete Freund’s adjuvant was used in subsequent injections. Ten millilitre of blood was collected from the rabbit ear vein every month for serum extraction. To produce purified M4P, 0.5 ml beads containing GST protein was used to eliminate nonspecific antibodies against GST. The serum was then incubated with PVDF membrane containing 1 mg immobilised antigen overnight at 4 °C before affinity-purified with an IgG elution buffer (21004, Thermo Fisher Scientific, MA, USA). The purified M4P antibody was quantified and diluted to 1 μg/μl for experiment.
Animal model and study design
This study was approved and conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of the National Neuroscience Institute, Singapore. All experiments were performed according to Stroke Therapy Academic Industry Roundtable (STAIR) recommendations . Allocation of animal treatment was randomised by throwing a dice. The middle cerebral artery occlusion (MCAO) method has been described previously .
The animals were housed with temperature maintained at around 23 °C and 12/12-h light/dark cycle was set. Pelleted food and water were available for the animals. The animals were monitored on a daily basis. All researchers involved in the study were blinded to the intervention. The infarct volume calculation at day 1 post operation was a predetermined primary end point, and the completion of functional study was the secondary end point. The study would be terminated if the mortality rate at primary end point is more than 60%. The mortality rates from different treatments were calculated and compared. Animal death during operation was not counted for mortality analysis. Animal exclusion criteria include (1) cerebral blood flow reduction post occlusion was < 50%; (2) rats without motor functional deficit assessed by Rotarod test (≥ 100% of baseline); (3) rats died during the observation periods.
In brief, male Wistar rats weighing approximately 250–280 g were anesthetised with ketamine (75 mg/kg) and xylazine (10 mg/kg) intraperitoneally. Relative regional cerebral blood flow of the animals was monitored by a Laser-Doppler flowmetry (moorVMS-LDF2™, Moor Instruments Inc., DE, USA). Heart rate, blood pressure, and rectal temperature were monitored using a data acquisition system PowerLab 4/35 from AD Instruments. The body temperature was maintained at 37 °C ± 0.5 °C with a warm pad throughout the procedure. The left common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) were dissected out. A silicon-coated filament (0.37 mm, Cat #403756PK10, Doccol Corp, CA, USA) was introduced into the left ICA through ECA. Cerebral blood flow of the animals was monitored by a Laser-Doppler flowmetry (moorVMS-LDF2™, Moor Instruments Inc., DE, USA). Animals with ≤ 50% cerebral blood flow reduction were excluded from the study. Reperfusion was achieved by removing the filament gently from the ECA at 3 h following occlusion. A single dose of 100 μg of M4P antibody  or control rabbit IgG (I5006, Sigma-Aldrich, MI, USA) was injected intravenously via tail vein at 2 h after occlusion (1 h before recanalization). For control permanent MCAO model, animals received similar operational procedure except for filament removal. For in vivo binding of M4P (Figs. 4a and 5a), MCAO was performed as per described. Two hours after occlusion, 100 μg of M4P antibody, control rabbit IgG, or 100 μl vehicle solution was injected intravenously. Three hours after occlusion (1 h after antibody injection), the rats were sacrificed and perfused with PBS to remove residual antibodies in the circulation. The brains were sectioned and fixed with 4% paraformaldehyde. Immunofluorescent staining using secondary antibody against rabbit IgG was performed to detect antibody extravasation.
Infarct volume measurement
Twenty-four hours after surgery, the animals were sacrificed, and the brains were collected with cerebellum and overlying membranes being removed. The brains were sectioned into 8 slices using a brain-sectioning block, each with 2 mm in thickness. The brain slices were incubated for 30 min in a 0.1% solution of 2,3,4-triphenyltetrazolium chloride (TTC) (T4375, Sigma-Aldrich, MI, USA) at 37 °C. The sections were scanned, and the infarct size was analysed using an image analyser system (HP Scanjet G3110, HP Inc, CA, USA). Calculation of oedema-corrected lesion was performed as described previously .
Rotarod (Ugo Basile, Gemonio, Italy) was used to evaluate motor functions post stroke. Before operation, the rats received 3 training trials with 15-min intervals for 5 consecutive days. The rotarod was set to accelerate from 4 to 80 rpm within 10 min. The mean duration of time that the animals remained on the device 1 day before MCAO was recorded as internal baseline control. At different time points following surgery, the mean duration of latency was recorded and compared.
Cell transfection and cell death, immunofluorescent staining, western blot, and surface biotinylation
For cell culture, HEK 293 cells were seeded on coated coverslips in 35 mm petri dish. Mouse TRPM4 (pIRES-EGFP-TRPM4) was transiently expressed using the calcium phosphate transfection method. Twenty-four hours after transfection, M4P or control rabbit IgG was added into the culture medium to a concentration of 0.26 μg/ml. After incubation for 30 min, 3 h, or 2 days, the cells were fixed with 4% paraformaldehyde. After washing three times with washing buffer (0.2% Triton X-100 phosphate-buffered saline), the samples were incubated with secondary antibody against rabbit IgG (Alexa Fluor 594 conjugated, Thermo Fisher Scientific, MA, USA) for 1 h before being mounted with FluorSaveTM reagent (345789, Millipore, MA, USA). For rat brain staining, the brains were harvested and sectioned at 10 μm in thickness. Following fixation with 4% paraformaldehyde, the brain slice was incubated in 100 μl blocking serum (10% fetal bovine serum in 0.2% PBST) for 1 h. Primary antibodies include M4P (rabbit, 10 ng/μl), anti-NeuN (MAB377, Millipore, MA, USA, 1:250), anti-GFAP (IF03L, Millipore, MA, USA, 1:200), and anti-vWF (AB7356, Millipore, MA, USA, 1:200). Secondary antibodies are conjugated with FITC or Alexa Fluor 594. Images were visualised by a confocal microscope (Fluoview BX61, Olympus, Tokyo, Japan). ImageJ was used to quantify the fluorescent intensity and vascular diameter. The average diameter was determined by using the shortest Feret diameter (Feret Min) as described previously . To quantify the fluorescence of M4P labelled vessels in rat brains, an outline was drawn around each vessel. Area, mean fluorescence, along with several adjunct background readings were measured using ImageJ. The total corrected fluorescence was calculated according to the formula: total corrected fluorescence density = (integrated fluorescence signal of selected vessels − area of selected vessels × mean fluorescence of background signal) / area of selected vessels [18, 19]. Identical acquisition conditions were used to capture images.
Cell death was determined using the Trypan blue exclusion method. TRPM4 transfected HEK 293 cells which were subjected to oxygen/glucose deprivation (OGD) for 24 h. Hypoxia was induced by culturing the cells in a hypoxic chamber with 1% O2 and 5% CO2 at 37 °C, and glucose was removed from the culture media. IgG or M4P was added to the cells at a concentration of 1.3 μg/ml before OGD induction.
For western blot on rat brains, tissues from ipsilateral and contralateral hemispheres were harvested. Infarct area which was negatively stained by TTC was excluded. For western blot on HEK 293 cells, the cells grown in 6-well petri dishes were transfected with 3 μg mouse pIRES-EGFP-TRPM4, TRPM5, or GFP vector. To perform western blot, 30 μg of total protein was resolved on 10% SDS-PAGE gels at 80 V, and electrophoretically transferred to PVDF membranes (1620177, Bio-Rad, CA, USA) at 100 V for 2 h at 4 °C. After blocking with StartingBlock (PBS) blocking buffer (37538, Thermo Fisher Scientific, MA, USA) for 1 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies: M4P (1:200), anti-GFP (12603500, Roche, Basel, Switzerland, 1:500), anti-TRPM5 (acc-045, Alomone, Jerusalem, Israel, 1:200), anti-Transferrin Receptor (TfR, 13–6800, Thermo Fisher Scientific, MA, USA, 1:1000), and anti-actin (A1978, Sigma-Aldrich, MI, USA, 1:5000). After washing away primary antibodies, the membranes were incubated with secondary antibodies (A4416, A4914, Sigma-Aldrich, MI, USA, 1:5000) for 1 h at room temperature. For western blot to detect antibody extravasation (Fig. 5b), secondary antibody against rabbit IgG was applied directly without incubation with primary antibodies. The primary antibodies were the control rabbit IgG or the polyclonal M4P injected into the rats. Primary and secondary antibodies were prepared in StartingBlock (PBS) blocking buffer with 0.05% Tween®20 (P7949, Sigma-Aldrich, MI, USA). Washing buffers contained 0.1% Tween®20 dissolved in PBS. Amersham ECL Western Blotting Analysis System (RPN2109, GE Healthcare, IL, USA) was used and the bands were visualised using medical X-ray processor (MXP-2000, KODAK, NY, USA). Quantification was done using ImageJ.
TRPM4 surface expression was characterised using an EZ-LinkTM Sulfo-NHS-Biotinylation Kit (Thermo Fisher Scientific, MA, USA) with slight modification . HEK 293 cells transfected with TRPM4 were incubated with control rabbit IgG or M4P of 1.3 μg/ml for 6 h. The cells were then treated with 0.25 mg/ml Biotin and shaken for 1 h at 4 °C. Unbound biotin was removed by incubation with quenching buffer for 20 min and washed with cold TBS. Protein concentration of cell lysates were measured with Pierce BCA Protein Assay Kit (23227, Thermo Fisher Scientific, MA, USA). Ten microlitre of cell lysates was kept for SDS-PAGE analysis. The remaining cell lysates were incubated with NeutrAvidin (Thermo Fisher Scientific, MA, USA) overnight at 4 °C to pull down the biotinylated surface proteins. The precipitates were boiled in 2× loading buffer to elute Avidin-bound for SDS-PAGE analysis.
Whole-cell patch clamp was used to measure TRPM4 currents in HEK293 cells grown in 24-well plates and transfected with 1 μg pIRES-EGFP-TRPM4 encoding mouse TRPM4 channel. TRPM4 currents were recorded at room temperature 24–48 h after transfection. Patch electrodes were pulled using a Flaming/Brown micropipette puller (P-1000, Sutter Instrument, CA, USA) and polished with a microforge (MF-200, WPI Inc. FL, USA). Whole-cell currents were recorded using a patch clamp amplifier (Multiclamp 700B equipped with Digidata 1440A, Molecular Devices, CA, USA). The bath solution contained (in millimole/litre) NaCl 140, CaCl2 2, KCl 2, MgCl2 1, glucose 20, and HEPES 20 at pH 7.4. The internal solution contained (in millimole/litre) CsCl 156, MgCl2 1, EGTA 10, and HEPES 10 at pH 7.2 adjusted with CsOH . Additional Ca2+ was added to get 7.4 μM free Ca2+ in the pipette solution, calculated using the program WEBMAXC v2.10. Rabbit IgG or M4P was added into bath solution at a concentration of 20.8 μg/ml for 30 min before recording. Ischemia/Hypoxia was induced by applying a bath solution containing 5 mM NaN3 and 10 mM 2-deoxyglucose (2-DG) continuously through a MicroFil (34 Gauge, WPI Inc. USA) around 10 μm away from the recording cells. The flow rate was 100 μl/min. The current–voltage relations were measured by applying voltage ramps for 250 ms from – 100 to + 100 mV at a holding potential of 0 mV. The sampling rate was 20 kHz and the filter setting was 1 KHz. Data were analysed using pClamp10, version10.2 (Molecular Devices, CA, USA).
Data are expressed as the mean ± s.e.m. Statistical analyses were performed using GraphPad Prism version 6.0. Two-tailed unpaired student’s t test was used to compare two means. One-way ANOVA with Bonferroni’s multiple comparison test was used to compare ≥ 3 means. Two-way ANOVA with Bonferroni’s multiple comparison test was used to analyse motor functions and time-dependent membrane capacitance change. Mortality rates were compared using Fisher exact probability test.
Generation and characterisation of TRPM4 blocking antibody M4P
TRPM4 blocking antibody M4P was designed to bind to a region close to the channel pore (Fig. 1a). Using western blot, M4P successfully detected TRPM4 channel in transfected HEK 293 cells (Fig. 1b). To examine whether M4P could bind to TRPM4 channel on the surface of live cells, HEK 293 cells transfected with TRPM4 were cultured with M4P or control rabbit IgG for 30 min, 3 h, and 2 days. The cells were then fixed and a secondary antibody against rabbit IgG was applied (M4P is a rabbit polyclonal antibody). As shown in Fig. 1c, M4P could be identified on the surface of HEK 293 cells. As the incubation time increased, more cytosolic staining was observed, suggesting a translocation of M4P from cell membrane to cytosol. No surface staining was observed in cells incubated with control IgG or secondary antibody respectively (Fig. 2 in the online-only Data Supplement). To study whether the M4P treatment could regulate the expression of TRPM4 on cell membrane, surface biotinylation assay was performed. After 6 h incubation, the total TRPM4 level was not changed, whereas M4P treatment downregulated surface TRPM4 (Fig. 1d and Fig. 3a in the online-only Data Supplement). It has been reported that TRPM4 can be glycosylated , and the glycosylation site is located within the epitope for M4P binding (Fig. 1a, 1b in the online-only Data Supplement). We therefore compared the ratio of the fully glycosylated TRPM4 fractions with the core glycosylated TRPM4 fractions and found no change in the ratio by M4P or control IgG treatments (Fig. 3 in the online-only Data Supplement).
We further examined whether M4P could bind to the paralogous TRPM5 channel. TRPM4 and TRPM5 share only 24% homology in the 28-amino acid sequence recognised by M4P (Fig. 4a in the online-only Data Supplement). In HEK 293 cells transfected with TRPM5, M4P could not bind to TRPM5 channel as shown by immunocytochemical and western blot methods (Fig. 4b, 4c in the online-only Data Supplement), suggesting that M4P does not cross react with TRPM5.
M4P inhibits TRPM4 current and protects transfected HEK 293 cells from hypoxia
Detection of TRPM4 channel by M4P in ischemic stroke
M4P attenuates cerebral injury in ischemia reperfusion
M4P protects cerebral vasculature in ischemia reperfusion
Recently, TRP channels have attracted attention as potential therapeutic targets for stroke . Here, we generated and characterised a TRPM4-specific blocking antibody M4P, targeting a region within the third extracellular domain of TRPM4 channel . As the antibody-binding motif is located extracellularly, M4P has easy access to membrane TRPM4 channels. The binding of M4P to TRPM4 channel was validated by both in vitro and in vivo studies. M4P was shown to inhibit TRPM4 activity via two mechanisms. The first is to inhibit the channel directly which could be more potent under hypoxic conditions, because ATP depletion can enhance TRPM4 activity . Secondly, incubation with M4P downregulated TRPM4 expression on cell surface, most likely via a mechanism seen in therapeutic antibodies in cancer treatment , where the formation of receptor-antibody complex induces endocytosis and subsequent protein degradation. The results from both in vitro data using cultured cells and in vivo data in stroke model indicated that M4P application could inhibit the expression of TRPM4. Importantly, as TRPM4 upregulation under hypoxia is known to cause oncotic cell death , our data strongly suggest that M4P incubation alleviates cell swelling under hypoxic conditions and reduces cell death.
Therapeutic antibodies targeting various receptors have been reported for stroke management . We employed stroke reperfusion model to evaluate the efficacy of M4P, antibodies were delivered 1 h before filament withdrawal, mimicking clinical scenario. One challenge for this study is that whether the antibody given before recanalization could travel to the hypoxic brain region where the blood vessel is blocked. Our results showed that M4P accumulated inside the vessels within the infarct area, possibly via collateral circulation, and bind to the endothelial TRPM4 where the channel is upregulated as early as 2 h after stroke .
It has been reported previously that 3 h occlusion resulted in a maximum infarct formation, and the occlusion time longer than 3 h did not increase infarct volume . In our 3-h stroke reperfusion model, M4P application could reduce infarct formation, accompanied with lower mortality rate and improvement of functional outcome. The results from Evans blue, antibody extravasation, and direct measurement of vascular diameter all strongly support that M4P treatment could improve vascular integrity, in line with our previous study using a similar stroke reperfusion model where cerebral oedema formation was ameliorated by TRPM4 siRNA .
Evidence showed that intravenous immunoglobulin (IVIG) treatment could modulate complement activation following stroke . But the IgG dose (0.1 mg/250 g) used in this study is much lower than the IVIG (> 0.5 g/kg). As M4P is a therapeutic antibody, it is possible that the immunomodulation effect may contribute to its therapeutic outcome in addition to blocking TRPM4. If a higher dosage is tolerable, increase of M4P dose could enhance its modulatory effect on the immune system during stroke.
Apart from vascular protection, M4P may yield neuroprotection via blocking neuronal TRPM4 channels. TRPM4 channel has been shown to be upregulated in neurons after stroke . In experimental autoimmune encephalomyelitis, TRPM4 inhibition protects neurons against neuroinflammation . Here, we demonstrated that M4P could bind to neurons close to the infarct core. In healthy brain, M4P is less likely to pass through BBB. However, as ischemic insult disrupts BBB, large molecules such as antibodies can enter brain parenchyma and block neuronal TRPM4 channels. Further study is needed to evaluate whether M4P yields a direct neuroprotection on neurons.
Besides stroke therapy, M4P can serve as a research tool to study TRPM4 channel. As the antigenic epitope for M4P production is unique to TRPM4 channel, M4P is more specific than other blockers. Glibenclamide inhibits TRPM4 via SUR1 which requires the surface expression of SUR1 protein with a high ratio to TRPM4 [4, 35]. Also, neurons express SUR1-KATP channel complex , which can be blocked by glibenclamide. Another TRPM4 blocker 9-phenanthrol interacts with TMEM16A channel, affecting vascular contraction . In heart, 9-phenanthrol non-selectively inhibits transient outward, rapid delayed rectifier, and inward rectifier K+ currents . Furthermore, as a member of polycyclic aromatic hydrocarbons, the toxicity of 9-phenanthrol remains a concern for in vivo usage.
In conclusion, we have developed a TRPM4-specific blocking antibody that can alleviate stroke injury during reperfusion. This strategy could avoid contraindications arising from other TRPM4 blockers. A future humanised TRPM4 blocking antibody could be a potential therapy for stroke in humans.
All authors contributed to the study conception and design. SWL and YG prepared the reagents. BC and GN performed the surgery. SWL and YG carried out functional and biochemical studies. SW performed electrophysiological experiments. BC, SWL, YG, SW, and PL analysed the data.
This work was supported by grants NMRC/OFIRG/0070/2018 and NMRC/CIRG/1425/2015 from the Singapore Ministry of Health’s National Medical Research Council.
Compliance with ethical standards
This study was approved and conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of the National Neuroscience Institute, Singapore.
Conflict of interest
The authors declare that they have no conflict of interest.
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