Activation of endothelial TRPM2 exacerbates blood–brain barrier degradation in ischemic stroke

Abstract

Aims

Damage of the blood–brain barrier (BBB) is a hallmark of brain injury during the early stages of ischemic stroke. The subsequent endothelial hyperpermeability drives the initial pathological changes and aggravates neuronal death. Transient receptor potential melastatin 2 (TRPM2) is a Ca²⁺-permeable nonselective cation channel activated by oxidative stress. However, whether TRPM2 is involved in BBB degradation during ischemic stroke remains unknown. We aimed to investigate the role of TRPM2 in BBB degradation during ischemic stroke and the underlying molecular mechanisms.

Methods and results

Specific deletion of Trpm2 in endothelial cells using Cdh5 Cre produces a potent protective effect against brain injury in mice subjected to middle cerebral artery occlusion (MCAO), which is characterized by reduced infarction size, mitigated plasma extravasation, suppressed immune cell invasion, and inhibited oxidative stress. In vitro experiments using cultured cerebral endothelial cells (CECs) demonstrated that either Trpm2 deletion or inhibition of TRPM2 activation attenuates oxidative stress, Ca²⁺ overload, and endothelial hyperpermeability induced by oxygen–glucose deprivation (OGD) and CD36 ligand thrombospondin-1 (TSP1). In transfected HEK293T cells, OGD and TSP1 activate TRPM2 in a CD36-dependent manner. Noticeably, in cultured CECs, deleting Trpm2 or inhibiting TRPM2 activation also suppresses the activation of CD36 and cellular dysfunction induced by OGD or TSP1.

Conclusions

In conclusion, our data reveal a novel molecular mechanism in which TRPM2 and CD36 promote the activation of each other, which exacerbates endothelial dysfunction during ischemic stroke. Our study suggests that TRPM2 in endothelial cells is a promising target for developing more effective and safer therapies for ischemic stroke.

Graphical Abstract

Graphical Abstract.

TRPM2-mediated Ca²⁺ signaling is essential for CD36-induced endothelial dysfunction and tight junction degradation. The activation of CD36 and TRPM2 forms a positive feedback loop that promotes blood–brain barrier degradation during ischemic stroke.

Time of primary review: 56 days

Translational perspective.

• Endothelial-specific Trpm2 deletion produces strong protective effects against ischemic stroke by preserving blood–brain barrier (BBB) integrity, inhibiting plasma extravasation, and inhibiting immune cell infiltration.

• Thrombospodin-1 (TSP1) activates endothelial TRPM2 in a CD36-dependent manner, leading to hyperpermeability of the BBB, therefore exacerbating ischemic stroke.

• TRPM2 activation is also required for CD36-induced endothelial dysfunction and subsequent tight junction degradation when exposed to oxygen–glucose deprivation (OGD) and TSP1.

• The TSP1–CD36–TRPM2 axis contributes to BBB degradation during ischemic stroke.

• Our data establish TRPM2 activation by TSP-1 as a new mechanism for endothelial hyperpermeability. Hence, TRPM2 in endothelial cells presents itself as a promising therapeutic target for mitigating BBB leakage during ischemic stroke.

1. Introduction

Ischemic stroke is a major health issue worldwide, with over 10 million new cases happening every year.¹ The central pathological feature of ischemic stroke is neuronal death.² Unfortunately, drugs mitigating neuronal death, such as N-methyl-D-aspartate (NMDA) receptor antagonists, ion channel blockers, caspase inhibitors, and neurotrophic factors, all failed to show protective effects in patients with ischemic stroke.^3,4 The failure of directly targeting neurons in the treatment of ischemic stroke patients has led to the consensus that targeting non-neuron cells, which contribute to neuronal death during ischemic stroke, may provide better therapeutic outcome.^5,6 Given the complexity of ischemic stroke, all the cells distal to the occlusion site, such as endothelial cells in the neurovascular unit (NVU), are influenced and involved in the progression of ischemic brain injury.

NVU is composed of endothelial cells, pericytes, basal lamina, smooth muscle cells, surrounding astrocytes, and neurons.⁷ Anatomically, endothelial cells are the core component of NVU.⁸ Compared to peripheral endothelial cells, a prominent feature of cerebral endothelial cells (CECs) are the widely linked tight junctions,⁹ which are critical for the formation of the blood–brain barrier (BBB). During ischemic stroke, tight junctions between endothelial cells are disrupted, and BBB is degenerated, which leads to fluid imbalance and immune cell invasion into the brain.^10–12 Both cerebral edema and inflammatory infiltration aggravate neuronal death and increase the mortality of patients with ischemic stroke.^11,13 Therefore, preserving BBB integrity by mitigating endothelial dysfunction should protect the brain against ischemic stroke. However, the molecular mechanisms causing endothelial dysfunction during ischemic stroke are poorly understood.¹⁴

CD36 in endothelial cells has been found to be involved in endothelial dysfunction during ischemic stroke.¹⁵ Interestingly, recent single-cell RNA sequencing works have identified a distinct endothelial cell population with abundant CD36 expression.¹⁶ CD36 is a member of the class B scavenger receptor family. This membrane protein has a large extracellular domain, which binds to different ligands, such as oxidized low-density lipoprotein (ox-LDL), thrombospondins (TSPs), and fibrillar β amyloid (fAβ).¹⁷ Ligand binding to CD36 triggers downstream signaling cascades, including activation of Fyn, JNK, and p38, all of which result in oxidative stress and cellular dysfunction in a variety of pathological conditions, including Alzheimer’s disease and ischemic stroke.^18–21 However, the underlying mechanisms regulating the activation of CD36 signaling remain unclear.¹⁵

TRPM2 is an oxidative stress sensitive and Ca²⁺-permeable nonselective cation channel activated by intracellular Ca²⁺ and ADP-ribose (ADPR). ADPR can be generated during oxidative stress and promotes cellular damage by activating TRPM2.²² TRPM2 is ubiquitously expressed in various cell types including endothelial cells and macrophages and is most abundantly expressed in the central nervous system.²³ Global Trpm2 knockout in mice has been shown to alleviate ischemic stroke, but the mechanisms underlying this phenomenon are not fully understood.^24–26 Knockdown or inhibition of TRPM2 attenuates the enhanced endothelial permeability induced by H₂O₂ in isolated pulmonary artery endothelial cells in vitro,²⁴ and Trpm2 deletion in endothelial cells reduced neutrophil transmigration in the lung in response to lipopolysaccharide (LPS) challenge.²⁷ However, it is unknown whether TRPM2 in CECs influences endothelial permeability during ischemic stroke. As mentioned earlier, endothelial cells in the central nervous system are significantly different from peripheral endothelial cells. Thus, we propose that TRPM2 promotes endothelial dysfunction and BBB degradation in ischemic stroke.

In this study, we found that selective deletion of Trpm2 in endothelial cells protects mice against ischemic stroke. Trpm2 deletion inhibits oxidative stress and prevents tight junction degradation between CECs and reduces the infiltration of immune cells into the brain after ischemic stroke. Mechanistically, we revealed that TRPM2 can be activated by CD36 ligand TSP1. Moreover, TRPM2 is required for the activation of CD36 signaling cascades in CECs induced by oxygen–glucose depletion (OGD) or TSP1. Our results establish an inter-dependent regulation of TRPM2 and CD36 in causing endothelial hyperpermeability during ischemic stroke, suggesting that targeting TRPM2 in CECs may represent a more effective approach to mitigate ischemic brain injury.

2. Methods

Animal experiments were approved by the animal care committees of University of Connecticut School of Medicine. All experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of University of Connecticut School of Medicine (animal protocol: AP-200135-0723) and were conducted in accordance with the US National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Mice were euthanized based on IACUC-approved protocols.

For details on materials regarding the origin of animals, plasmids, and antibodies used in our study, please see the Supplementary material online. Furthermore, detailed information regarding surgery procedures, cell cultures, plasmid transfections, whole-cell current recordings, immunoblot analysis, immunofluorescence staining, and Ca²⁺ and mitochondrial imaging techniques can also be found in the Supplementary material online.

3. Results

3.1. Endothelial cell–specific Trpm2 deletion attenuates ischemic stroke

To examine the role of endothelial TRPM2 in ischemic stroke, Trpm2 was selectively knocked out in endothelial cells using Cdh5-cre mice (see Supplementary material online, Figure S1A), which wasconfirmed by western blot (WB) (see Supplementary material online, Figure S1B and C) and whole-cell current recording (see Supplementary material online, Figure S1D and E). Importantly, TRPM2 knockout did not influence the expression of other oxidative stress-related TRP channels, TRPV1, TRPA1, TRPM4, TRPM7, and TRPM8, in endothelial cells (see Supplementary material online, Figure S1F). The endothelial cell–specific Trpm2 knockout (Cre⁺, Trpm2^fl/fl) mice are designated as eM2KO, while the Cre⁻ littermates (Cre⁻, Trpm2^fl/fl) are designated as WT hereafter.

Infarct volume was examined by 2,3,5-triphenyltetrazolium chloride (TTC) staining 24 h after MCAO (Figure 1A). The successful occlusion of middle cerebral artery was confirmed by reduced blood flow during occlusion measured by laser Doppler blood FlowMeter (Figure 1B; Supplementary material online, Figure S1G). Mice with Trpm2 deletion in endothelial cells (eM2KO) exhibited smaller infarction size (18.9 ± 1.4% in eM2KO vs. 34.4 ± 3.5% in WT) and improved neurological performance (1.4 ± 0.2 in eM2KO vs. 2.6 ± 0.3 in WT) (Figure 1A, C, and D). Increase of plasma extravasation examined by Evans blue assay (Figure 1E and F) after MCAO was attenuated in eM2KO mice (0.3 ± 0.1 in eM2KO vs. 0.7 ± 0.1 in WT).

Figure 1.

Endothelial cell–specific Trpm2 deletion alleviates ischemic stroke. (A–F) Evaluation 24 h after MCAO. (A) TTC staining of brain slices (1 mm). (B) Blood flow changes. Reduction of CBF for over 85% indicates successful MCAO. (C, D) Mean infarct volume and average ND score after MCAO from WT (n = 10) and eM2KO mice (n = 12). (E) Evans blue assay of whole brain. (F) Mean Evans blue absorption at 620 nm after MCAO from WT (n = 8) and eM2KO mice (n = 8). (G–J) Evaluation 6 h after MCAO (n = 6, 7). (G) Evans blue assay and TTC staining. (H) Mean infarct volume. (I) Mean Evans blue absorption. (J) Average ND score. (K–N) Evaluation 7 days after MCAO (n = 7, 7). (K) Evans blue assay and TTC staining. (L) Mean infarct volume. (M) Quantification of Evans blue assay. (N) Average ND score. (O) Representative images of cerebral vasculature imaging. (P) Quantification of superior sagittal sinus (SSS) diameter (n = 8, 9). (Q) Representative images of cerebral perfusion. (R) Quantification of SSS perfusion (n = 8, 9). (S) Representative images of CD31 staining of brain slices (5 µm). (T) Quantification of CD31 density (n = 6, 6). (U) Representative images of tomato lectin microvasculature staining of brain slices (50 µm). (V) Quantification of capillary density (n = 8, 9). (W) Quantification of capillary diameter (n = 8, 9) (unpaired t-test with Welch’s correction).

We also evaluated the early protective effects of eM2KO after ischemic stroke. We found that eM2KO reduced brain infarction as well as Evans blue leakage as early as 6 h after MCAO (Figure 1G–I) and prevented the compromise of neurobehavior functions (Figure 1J).To examine whether eM2KO produces a long-term protective effect against ischemic stroke, we performed TTC staining and Evans blue assay 7 days after the reperfusion of MCAO and observed reduced brain infarction and BBB leakage in eM2KO group (Figure 1K–M). Moreover, we evaluated the brain function at Days 1, 3, and 7 by neurological deficit (ND) score and rotarod assay. We found that eM2KO prevented the deterioration of neurological performance after MCAO (Figure 1N; Supplementary material online, Figure S1H). These results indicate that endothelial cell–specific TRPM2 knockout attenuates ischemic stroke in mice.

Ischemic stroke in human patients usually involves multiple risk factors, including hypercholesterolemia.²⁸ We sought to understand whether eM2KO still produces a protective effect in model mice with at least one risk factor. We fed mice with high-fat diet (HFD) to induce hypercholesterolemia. After a 4-month HFD treatment, all mice developed obesity and dyslipidemia, and there is no difference in the body weight (see Supplementary material online, Figure S1I) and serum cholesterol level (see Supplementary material online, Figure S1J) between WT and eM2KO mice. We found that after MCAO, eM2KO mice still exhibited smaller infarct size (see Supplementary material online, Figure S1K and L) and preserved neurobehavior performance (see Supplementary material online, Figure S1M). These data further confirm the potential translational values of our findings in human patients.

TRPM2 was previously shown to promote angiogenesis in a hindlimb ischemia model.²⁹ Thus, we examined whether eM2KO influence vasculature and cerebral blood flow (CBF). We used laser speckle imaging system to monitor the real-time in vivo cerebral circulation.³⁰ We found that eM2KO did not cause a significant change in vessel diameter (Figure 1O and P) or CBF (Figure 1Q and R). Moreover, there was no difference of the endothelial cell density (Figure 1S and T) and capillary diameter/density between WT and eM2KO mice, suggesting that TRPM2 knockout does not affect the basal cerebral microcirculation (Figure 1U–W). Our data indicate that TRPM2 does not influence the vasculature development in the brain under normal physiological conditions.

Endothelial cells are the core component of the NVU/BBB, and the loss of tight junctions between endothelial cells is one of the hallmark events at the initial stage of ischemic stroke.¹⁴ As occludin is a crucial component of the tight junction,³¹ we investigated whether occludin function was impacted. After MCAO, the expression of occludin was decreased in WT mice, whereas this decrease was inhibited in eM2KO mice (see Supplementary material online, Figure S2A and B). Also, TUNEL staining revealed that the apoptosis of endothelial cells after MCAO was attenuated by eM2KO (see Supplementary material online, Figure S2C and D).

3.2. eM2KO alleviates post-stroke inflammatory infiltration

Immune cell infiltration follows BBB degradation and aggravates brain injury. We prepared single-cell suspension of the brain following a well-established protocol and performed flow cytometry analysis.³² We used DAPI to exclude the dead cells in the single-cell gate (Figure 2A). Then, we used CD45 to identify the immune cell populations, in which the CD45^high population is blood-derived infiltrated leukocytes, and the CD45^medium population is the endogenous microglia³² (Figure 2B–E). Based on the cell size, CD45^high leukocytes were divided into two populations, myeloid cells and lymphocytic cells. Myeloid cells were divided into neutrophils and monocytes based on the neutrophil marker Ly6G, and lymphocytic cells were divided into B cells, T cells, and NK cells based on the B cell marker CD19 and the T cell marker CD3e (Figure 2B–E). Ly6C was used to identify the activation status of monocytes and microglia (Figure 2B–E).

Figure 2.

Trpm2 deletion attenuates post-stroke immune cell invasion. (A) Gating strategy of alive cells from single-cell suspension after brain digestion. Single cells were identified from the scatter gate, which were further divided into living cells and dead cells based on DAPI intensity. (B–E) Representative flow cytometry analysis of immune cell populations. Among alive cells, CD45 was used to identify nonimmune cells (CD45⁻), microglia (CD45^medium), and leukocytes (CD45^high). Microglia were divided into two populations based on Ly6C expression. Leukocytes were divided into myeloid and lymphocytic cells based on cell size. Lymphocytes were divided into B cells, T cells, and NK cells based on CD19 and CD3e. Myeloid cells were divided into neutrophils and monocytes based on Ly6G. Monocytes were further divided into two populations based on Ly6C expression. Quantification of CD45^high leukocytes (F), CD45^medium microglia (G), CD3e⁺ T cells (H), CD3e⁻CD19⁻ NK cells (I), Ly6G⁺ neutrophils (J), and CD11b⁺Ly6G⁻ monocytes (K) (n = 9, 8) (unpaired t-test with Welch’s correction).

We found that eM2KO did not alter the immune cell populations in the contralateral hemisphere (control side) 24 h after MCAO but inhibited the increase of blood leukocyte infiltration into the ipsilateral hemisphere (infarction side) (Figure 2D–F). The composite roles of T cells in ischemic stroke remain controversial, but the overall effects of T cells in the early stages of ischemic stroke is proinflammatory and can aggregate tissue injury.³³ We found that compared to the WT mice, T cell infiltration after MCAO was inhibited by eM2KO (Figure 2D, E, and H). We barely detected any B cells in the brains (Figure 2B–E), indicating that our isolation is successful as there is no contamination of B cells from the blood or lymph nodes. NK cell infiltration also promotes ischemic brain injury.³⁴ We found that the increased infiltration of CD3e⁻CD19⁻ lymphocytic cells in WT mice after MCAO was reduced in eM2KO mice (Figure 2D, E, and I), suggesting that NK cell invasion was attenuated.

Neutrophils are the first immune cells to arrive during an ischemic stroke within hours and aggravate the brain injury.³⁵ We found that the enhanced infiltration of CD11b⁺Ly6G⁺ neutrophils after MCAO in WT mice was attenuated in eM2KO mice (Figure 2D, E, and J), which was confirmed by myeloperoxidase (MPO, a neutrophil marker) staining of the penumbra (see Supplementary material online, Figure S2E and F). Also, WB analysis showed that the increased expression of MPO in the brain after MCAO was attenuated by eM2KO (see Supplementary material online, Figure S2I and J). After ischemic stroke, monocyte/macrophage infiltration happens within 12 h and causes a long-term inflammatory response.³⁵ We found that infiltration of CD11b⁺Ly6G⁻ monocytes after MCAO was suppressed in eM2KO mice (Figure 2D, E, and K), which was also examined by F4/80 staining (see Supplementary material online, Figure S2G and H) and WB analysis of CD11b (see Supplementary material online, Figure S2I and J).

Not surprisingly, compared with WT mice, in eM2KO mice, we observed an inhibition of the mRNA expression of inflammatory chemokines Ccl2 (Mcp1), Cxcl1, and Cxcl2 (see Supplementary material online, Figure S2K), as well as the reduced protein expression of MCP1 (see Supplementary material online, Figure S2L and M). In summary, the above results suggest that Trpm2 deletion in endothelial cells protects against ischemic brain injury by mitigating BBB leakage and inflammatory infiltration.

3.3. Trpm2 deletion prevents CEC hyperpermeability induced by OGD

Besides endothelial dysfunction, many other factors, such as immune cell invasion and glutamate excitotoxicity, also contribute to the disruption of BBB during ischemic stroke.⁷ Therefore, it is necessary to examine the role of TRPM2 in endothelial permeability by in vitro experiments using isolated primary cells. CECs were isolated based on a well-developed protocol.³⁶ We used OGD to treat CECs, as OGD can better mimic in vivo ischemic injury condition compared with H₂O₂ treatment.³⁷ We found that a 4- or 8-h OGD markedly down-regulated the mRNA expression of tight junction markers Zo1, Claudin5, and Occludin (see Supplementary material online, Figure S3A) and reduced the protein expression of occludin (Figure 3A and B) in WT CECs but not in TRPM2-KO CECs (see Supplementary material online, Figure S3A; Figure 3A and B). As the 8-h OGD treatment caused an unnecessary increase of CEC death, we chose to use 4 h as our treatment duration. Interestingly, different to the increased expression of proinflammatory chemokines in vivo, TRPM2 knockout did not inhibit the OGD-induced up-regulation of Ccl2 and Cxcl1 in CECs in vitro (see Supplementary material online, Figure S3D), suggesting that endothelial cells may not be the major source of chemokine production during ischemic stroke.

Figure 3.

Trpm2 deletion prevents OGD-induced cerebral endothelial hyperpermeability. (A, B) Trpm2 deletion preserved occludin expression. Representative WB analysis of the expression of occludin in CECs 2, 4, and 8 h after OGD (n = 5/group). (C) In vitro leakage assay as detailed in Supplementary material online, Figure S3C. Quantification of Evans blue absorption from lower chamber at 610 nm (n = 6/group). (D, E) In vitro macrophage infiltration assay as detailed in Supplementary material online, Figure S3D. F4/80 and CD80 staining of macrophages in lower chamber was performed as in (D). (E) Quantification of the number of infiltrated macrophages under ×10 field (n = 6/group). (F, G) WB analysis of the expression of pNOS3, NOS3, and NOS2 in the brain 24 h after sham surgery or MCAO (n = 6/group). (H, I) WB analysis of the expression of pNOS3, NOS3, and NOS2 in isolated CECs (n = 6/group). (J, K) Trpm2 deletion inhibited ROS production in CECs. (J) Rh123 real-time imaging before and 15 min after OGD in CECs. (K) Quantification of changes of Rh123 fluorescence 15 min after OGD. WT (n = 62 for OGD, n = 46 for control) and M2KO (n = 40 for OGD, n = 39 for control) CECs were from four dishes of cultured cells isolated from three mice in each group. (L–N) Trpm2 deletion inhibited Ca²⁺ overload in CECs. (L) Representative images at 0, 15, and 30 min during OGD. (M) Averaged traces from 10 CECs. (N) Quantification of Fura-2 changes 30 min after OGD. WT (n = 36 for OGD, n = 36 for control) and M2KO (n = 53 for OGD, n = 45 for control) CECs were from four dishes of cultured cells isolated from three mice in each group (unpaired t-test with Welch’s correction).

To evaluate endothelial permeability,³⁸ CECs were plated onto the upper chamber of transwell inserts with pore sizes of 0.4 µm. When endothelial cells completely covered the upper surface of inserts 3–5 days after seeding, either a 4-h OGD or control treatment was applied to the cells, and endothelial permeability was evaluated by adding Evans blue to the upper chamber (see Supplementary material online, Figure S3C). A marked leakage of Evans blue into the lower chamber was induced by OGD in CECs isolated from WT mice, but this leakage was inhibited in the CECs isolated from eM2KO (Figure 3C). These results suggest that deletion of Trpm2 inhibits the increase in endothelial permeability after OGD, which is consistent with the in vivo results showing the reduced leakage in MCAO brain in Trpm2 deletion mice (Figure 1).

We next used in vitro macrophage infiltration assay to further examine endothelial permeability to immune cells.³⁹ CECs were plated onto transwell inserts with pore sizes of 12 µM for macrophage migration. After endothelial cells were exposed to OGD or control treatment for 4 h, bone marrow–derived macrophages (BMDMs) were added into the upper chamber, while C5a was added into the lower chamber to promote macrophage infiltration. The infiltrated BMDMs on the 25 mm coverslips in the lower chamber were examined 12 h later by immunostaining with the macrophage marker F4/80 and the proinflammatory macrophage (M1) marker CD80 (see Supplementary material online, Figure S3D). We found that OGD treatment markedly increased the infiltration of macrophages into the lower chamber (Figure 3D and E), suggesting the degradation of tight junctions between CECs. Importantly, there were much fewer macrophages detected in the lower chamber in eM2KO group (Figure 3D and E), suggesting that Trpm2 deletion cells prevent tight junction degradation caused by OGD. These results recapitulate our in vivo data that Trpm2 deletion inhibits immune cell invasion after MCAO.

3.4. Trpm2 deletion inhibits OGD-induced cellular stress in CECs

Next, we sought to understand how Trpm2 deletion prevents endothelial dysfunction. Nitric oxide synthase 3 (NOS3), the endothelial cell–specific NOS (eNOS), produces nitric oxide (NO) during oxidative stress,⁴⁰ which can neutralize the over-produced reactive oxygen species (ROS), hence protecting CECs against ischemic injury.^40–42 Phosphorylation of NOS3 at Serine 1179 (pNOS3) increases NO production and minimizes ROS production,^40–42 whereas the ubiquitously expressed nitric oxide synthase 2 (NOS2), the inducible NOS (iNOS), promotes ROS production by causing NOS uncoupling during oxidative stress and increases CEC damage under ischemia.^40,43

Although there was no significant increase of NOS3 phosphorylation, we found that the expression of NOS3 was increased in brains from WT mice 24 h after MCAO, suggesting the activation of endogenous protection mechanism against oxidative stress in CECs (Figure 3F and G). In contrast, compared to WT mice brains, the brains from eM2KO mice after MCAO showed a higher level of NOS3 expression and a marked increase of NOS3 phosphorylation (Figure 3F and G). We also examined NOS3 phosphorylation in the brain from mice with neuron-specific Trpm2 deletion (nM2KO), which was found to protect mice from ischemic stroke in our previous work.⁴⁴ We found that, although there was a slightly higher increase of NOS3 expression in nM2KO after MCAO compared to WT mice, the observed increase of NOS3 phosphorylation in eM2KO mice was absent in nM2KO mice (see Supplementary material online, Figure S3E and F). This result suggests that the increased phosphorylation of NOS3 was not caused by mitigated tissue damage but was rather associated with the specific deletion of Trpm2 in endothelial cells, and that TRPM2 activation in endothelial cells after MCAO inhibits NOS3 phosphorylation. Additionally, the pathologically heightened expression of NOS2 after MCAO was inhibited in eM2KO mice (Figure 3F and G).

NOS3 was also reported to be expressed in some neuronal cells.⁴⁵ To further confirm that the change in NOS3 expression and phosphorylation in the brain (Figure 3F and G) was attributed to endothelial cells, we examined NOS expression in isolated CECs subjected to OGD. We found that there was an increase in NOS3 phosphorylation in M2KO CECs compared with WT CECs (Figure 3H and I). Moreover, the increased expression of NOS2 induced by OGD was inhibited in M2KO CECs (Figure 3H and I). The increased NOS3 phosphorylation and reduced NOS2 expression in M2KO CECs predict an increased NO production but a decreased ROS production in response to OGD treatment.^40,43

We next measured ROS production in CECs using Rhodamine-123 (Rh123), a commonly used dye for monitoring mitochondria membrane potential and ROS production.⁴⁶ Ischemia causes mitochondria membrane depolarization⁴⁷ and mitochondrial dysfunction,⁴⁸ which transform mitochondria into ROS production machines.^49,50 We found that OGD induced a marked increase of Rh123 fluorescence in WT CECs in 15 min, but this increase was inhibited in eM2KO CECs (Figure 3J and K), suggesting the preserved mitochondrial function and reduced ROS production in M2KO CECs.

One major cause of mitochondria depolarization and dysfunction is Ca²⁺ overload.⁷ Thus, we measured intracellular Ca²⁺ concentration using real-time ratio Ca²⁺ imaging. OGD induced a sustained increase of intracellular Ca²⁺ in WT CECs, but this increase was suppressed in eM2KO CECs (Figure 3L–N). In summary, we found that M2KO preserved mitochondria function, inhibited ROS production, and mitigated Ca²⁺ overload in CECs subjected to OGD.

3.5. Trpm2 deletion inhibits CD36 activation by OGD and TSP1 in CECs

We subsequently investigated the underlying molecular mechanisms of M2KO-mediated protection against endothelial dysfunction and hyperpermeability. We found that OGD markedly increased the expression of TRPM2 by 4.24-fold (see Supplementary material online, Figure S4A and B), which is consistent with our previous report that the expression of TPRM2 in the brain is substantially up-regulated after MCAO.⁴⁴ Another member of the TRPM family, TRPM4, was also found to promote endothelial dysfunction during ischemic stroke.⁵¹ We found that the basal expression level of TRPM4 appeared to be much higher than TRPM2 (see Supplementary material online, Figure S3H and I). Similar to TRPM2, the expression of TRPM4 was also up-regulated by OGD, but this increase (1.48-fold) is smaller than TRPM2 (see Supplementary material online, Figure S4A and B). After a 4-h OGD treatment, we also recorded much greater TRPM2 current in CECs (see Supplementary material online, Figure S3C and D). Our results suggest that although TRPM4 has a higher expression level than TRPM2 in CECs under physiological conditions, TRPM2 expression can be substantially enhanced to a further extent by a pathological stimulus.

Endothelial hyperpermeability after ischemic stroke can be induced by various signaling pathways including CD36.⁵² We found that CD36 expression in the brain was increased by MCAO, but this increase was inhibited by Trpm2 deletion (see Supplementary material online, Figure S4E and F). Considering immune cells have a high expression of CD36, we performed OGD using isolated CECs to better examine the role of CD36 in endothelial dysfunction. Our results show that CD36 expression was increased in WT CECs by OGD but not in eM2KO CECs (Figure 4A and B). Importantly, the basal expression level of CD36 in the brain and CECs is not significantly altered by eM2KO (Figure 4A and B; Supplementary material online, Figure S4E and F).

Figure 4.

Trpm2 deletion suppresses CD36 signaling activation. WB analysis of the expression of CD36, pFyn, Fyn, pJNK, JNK, pp38, and p38 in CECs subjected to OGD (A, B) and TSP1 (C, D) treatment (n = 6/group). (E, F) WB analysis of the expression of pNOS3, NOS3, and NOS2 in CECs (n = 6/group). (G) Rh123 imaging before and 10 min after TSP1 perfusion in CECs. (H) Quantification of Rh123 changes. WT (n = 32 for OGD, n = 44 for control) and M2KO (n = 43 for OGD, n = 33 for control) CECs were from four dishes isolated from three mice in each group. (I) Representative images at 0, 10, and 20 min during TSP1 perfusion. (J) Averaged traces were from 10 CECs. (K) Quantification of Fura-2 changes 20 min after TSP1 perfusion. WT (n = 31 for OGD, n = 62 for control) and M2KO (n = 32 for OGD, n = 40 for control) CECs were from four dishes isolated from three mice in each group. (L, M) WB analysis of the expression of occludin in CECs (n = 6/group). (N) Quantification of in vitro endothelial cell permeability test (as in Supplementary material online, Figure S3E). (O) In vitro macrophage infiltration test (as in Supplementary material online, Figure S3F) (unpaired t-test with Welch’s correction).

Activation of CD36 triggers downstream signaling of factors associated with oxidative stress, ROS production, and cell death, including Fyn, JNK, and p38.^17,53,54 We found that in WT CECs exposed to OGD, phosphorylation of Fyn, JNK, and p38 was markedly increased, whereas this increase was inhibited in eM2KO CECs (Figure 4A and B), suggesting that TRPM2 is critical for CD36 signaling activation induced by OGD. Since OGD can also cause many nonspecific cellular responses, we further examined the role of TRPM2 in CD36 activation in CECs using a CD36 ligand thrombospondin-1 (TSP1). TSP1 is a glycoprotein that can be secreted by endothelial cells, and the production of TSP1 in endothelial cells increases rapidly within hours following tissue injury or inflammation, leading to endothelial cell apoptosis during ischemic stroke.^55–57 Moreover, TSP1–CD36 signaling was found to increase endothelial hyperpermeability induced by vascular endothelial growth factor (VEGF).⁵⁸ We observed that a 4-h TSP1 treatment at 1 µg/mL induced a robust activation of CD36 signaling cascades in WT CECs, which was inhibited in M2KO CECs (Figure 4C and D).

Similar to OGD-induced responses, TSP1 increased NOS3 expression in both WT and eM2KO CECs but only promoted NOS3 phosphorylation in M2KO CECs (Figure 4E and F). This increased NOS2 expression induced by TSP1 was inhibited by M2KO (Figure 4E and F). TSP1 also induced a rapid increase of Rh123 fluorescence in WT endothelial cells, whereas this increase was blunted in M2KO CECs (Figure 4G and H), suggesting that TRPM2 promotes TSP1-mediated ROS production in mitochondria. Moreover, TSP1 perfusion induced a sustained increase of intracellular Ca²⁺ in WT CECs, which was inhibited by Trpm2 deletion (Figure 4I–K). These results indicate that TRPM2 is critical in magnifying TSP1-induced oxidative stress in CECs.

TSP1 treatment for 4 h caused a significant loss of occludin in WT CECs but not in M2KO CECs (Figure 4L and M). In in vitro permeability assay (see Supplementary material online, Figure S3E and F), TSP1 resulted in a moderate increase of Evans blue leakage (Figure 4N) compared to that caused by OGD (Figure 3C), and this increase was inhibited in M2KO CECs (Figure 4N). Similar to OGD-induced macrophage infiltration (Figure 3D and E), TSP1 also induced macrophage infiltration in WT CECs, which was inhibited in M2KO CECs (Figure 4O).

Ultimately, our data indicate that CD36 activation promotes endothelial hyperpermeability, and that TRPM2 is critical for the activation of CD36 signaling cascade during OGD.

3.6. Inhibiting TRPM2 suppresses CD36 activation in CECs

The observation of TRPM2-dependent CD36 activation makes us ask whether TRPM2 is activated during OGD and TSP1 treatment. We recorded TRPM2 current during OGD or TSP1 perfusion. The pipette solutions for TRPM2 recording contained 500 nM Ca²⁺ and 1 µM ADPR, which could not elicit TRPM2 current in HEK293 cells over-expressing TRPM2 without OGD treatment (Figure 5A and B). We used N-(p-amylcinnamoyl)anthranilic acid (ACA) to confirm TRPM2 current and NMDG to determine any involvement of leak current. We found that OGD treatment induced TRPM2 activation in HEK293T cells co-expressed with CD36 but not in HEK293T cells expressed with TRPM2 alone (Figure 5A–C). The OGD-induced activation of TRPM2 currents was inhibited by preincubation with a CD36-specific inhibitor, sulfosuccinimidyl oleate (sodium salt) (SSO) (Figure 5C; Supplementary material online, Figure S4G).

Figure 5.

Inhibiting the activation of TRPM2 suppresses the activation of CD36 signaling cascades in CECs. (A) Representative TRPM2 current traces elicited by OGD (upper trace: outward current at +100 mV; lower trace: inward current at +100 mV) in HEK293T cells transfected with CD36 and TRPM2. NMDG blocks inward current indicating the tightness of seal. ACA is a TRPM2 channel blocker. (B) Representative recording traces in HEK293T cells transfected with only TRPM2 during OGD. (C, H) Quantification of TRPM2 current amplitude in HEK293T cells transfected with CD36 and TRPM2 during OGD as in (C) and during TSP1 treatment as in (H) (n = 6/group). (D, E) Representative WB analysis of the expression of CD36, pFyn, Fyn, pJNK, JNK, pp38, and p38 in CECs (n = 5/group). (F) Representative TRPM2 current traces (upper trace: outward current at +100 mV; lower trace: inward current at +100 mV) in HEK293T cells transfected with CD36 and TRPM2 during TSP1 treatment. NMDG blocks inward current indicating the tightness of seal. ACA is a TRPM2 blocker. (G) Representative recording traces in HEK293T cells transfected with only TRPM2 during TSP1 treatment. (I, J) (I) Representative WB of the expression of CD36, pFyn, Fyn, pJNK, JNK, pp38, and p38 in CECs (n = 5/group) (two-way Welch ANOVA with Dunnett’s T3 test).

To understand how CD36 promotes TRPM2 activation by OGD, we investigated whether OGD and CD36 influence intracellular Ca²⁺ or ADPR, two critical factors required for the activation of TRPM2.⁵⁹ We used the PLC inhibitor U73122 to inhibit intracellular Ca²⁺ release and used a potent poly ADPR-ribose polymerase inhibitor, PJ34, to prevent ADPR production during OGD treatment. We found that preincubation of PJ34 and U73122 (in the presence of Ca²⁺ free extracellular recording solution) largely eliminated TRPM2 activation induced by OGD (Figure 5C; Supplementary material online, Figure S4G). As Trpm2 deletion markedly inhibited CD36 activation (Figure 4), we investigated whether inhibition of TRPM2 by ACA, PJ34, and U73122 also produces a similar effect. CECs incubated with ACA, PJ34, and U73122 displayed reduced activation of CD36 signaling after OGD treatment (Figure 5D and E).

TSP1 also activates TRPM2 current in HEK293T cells co-transfected with TRPM2 and CD36 (Figure 5F and H) but not in HEK293T cells transfected with TRPM2 alone (Figure 5G). Preincubation with SSO abolished the activation of TRPM2 by TSP1 (Figure 5H; Supplementary material online, Figure S4H). Similar to OGD, the activation of TRPM2 by TSP1 was eliminated when transfected cells were preincubated with ACA, PJ34, and U73122 (Figure 5H; Supplementary material online, Figure S4H). The activation of CD36 signaling cascades in CECs by TSP1 was blocked by inhibiting TRPM2 with ACA, PJ34, and U73122 (Figure 5H; Supplementary material online, Figure S4H).

The above data suggest that during OGD and TSP1 treatment, CD36 promotes TRPM2 activation by increasing ADPR production and intracellular Ca²⁺ concentration. Moreover, TRPM2-mediated Ca²⁺ signaling is also required for the activation of CD36 signaling cascades, which is similar to the critical role of TRPM2-mediated Ca²⁺ in many other cellular functions.⁶⁰

3.7. Inhibition of TRPM2 or CD36 prevents endothelial dysfunction induced by OGD and TSP1

Continuously, we examined whether inhibiting TRPM2 activation influences the phenotypic changes of CECs induced by OGD or TSP1 treatment. Preincubating CECs with SSO promoted NOS3 phosphorylation and inhibited the increase of NOS2 induced by either OGD (see Supplementary material online, Figure S5A and B) or TSP1 treatment (see Supplementary material online, Figure S5C and D). Using TRPM2 inhibitor ACA or inhibiting the activation of TRPM2 using PJ34 or U73122 produced similar effects on the expression of pNOS3 and NOS2 in CECs subjected to OGD (see Supplementary material online, Figure S5A and B) and TSP1 perfusion (see Supplementary material online, Figure S5C and D). SSO, ACA, PJ34, and U73122 also mitigated the mitochondrial depolarization and ROS production in CECs induced by either OGD (Figure 6A and C) or TSP1 treatment (Figure 6B and D). Moreover, the increase of intracellular Ca²⁺ in CECs induced by either OGD (Figure 6E and G) or TSP1 treatment (Figure 6F and H) was inhibited by SSO, ACA, PJ34, and U73122 preincubation. These results suggest that inhibiting TRPM2 produced a similar protective effect against oxidative stress in CECs induced by OGD as Trpm2 deletion.

Figure 6.

TRPM2 inhibition prevents oxidative stress and Ca²⁺ overload in endothelial cells after OGD or TSP1 treatment. (A–D) Representative picture of Rh123 imaging before and 15 min after OGD as in (A) and 10 min after TSP1 treatment as in (B) in isolated CECs with the treatment of DMSO, SSO, ACA, PJ34, and U73122. Quantification of changes of Rh123 fluorescence 15 min after OGD as in (C) and 5 min after TSP1 treatment as in (D) (n = 30–50/group). (E–H) Representative real-time Fura-2 Ca²⁺ imaging traces during OGD as in (E) and during TSP1 treatment as in (F) with the treatment of DMSO, SSO, ACA, PJ34, and U73122. The averaged traces were from 10 CECs. Quantification of Fura-2 fluorescence changes 30 min after OGD as in (G) and 20 min after TSP1 treatment as in (H) (n = 30–50/group) (two-way Welch ANOVA with Dunnett’s T3 test).

As NOS activity does not directly reflect NO generation, we directly measured NO production using DAF-FM in CECs subjected to OGD.⁶¹ The results showed that TRPM2 knockout or inhibition of TRPM2 activation preserved the compromised NO production in CECs induced by OGD (see Supplementary material online, Figure S6A and B) or TSP1 (see Supplementary material online, Figure S6C and D). Moreover, we used MitoSOX to directly evaluate mitochondrial ROS generation in CECs during OGD⁶² and found that the MitoSOX increase induced by OGD (see Supplementary material online, Figure S6E and F) or TSP1 (see Supplementary material online, Figure S6G and H) was suppressed by TRPM2 knockout or inhibition of TRPM2 activation.

Next, we tested whether the inhibition of TRPM2 preserves endothelial permeability. We found that SSO, ACA, PJ34, and U73122 inhibited the loss of occludin in WT CECs subjected to OGD (see Supplementary material online, Figure S7A and B) and TSP1 treatment (see Supplementary material online, Figure S7C and D). In in vitro endothelial permeability test, SSO, ACA, PJ34, and U73122 markedly decreased Evans blue leakage (see Supplementary material online, Figure S7E and F). Macrophage infiltration across CECs undergoing OGD (see Supplementary material online, Figure S7G and H) and TSP1 treatment (see Supplementary material online, Figure S7I and J) was also antagonized by SSO, ACA, PJ34, and U73122.

3.8. TRPM2 inhibition prevents post-stroke BBB leakage

ACA is a nonspecific TRPM2 inhibitor and can inhibit the activation of other channels, and SSO, PJ34, and U73122 did not directly inhibit TRPM2 activation. To further confirm the translational value of TRPM2 inhibition against endothelial dysfunction, we used a highly selective TRPM2 inhibitory peptide, TAT-M2. TAT-M2 works by blocking the binding of ADPR to TRPM2, and it has been shown to attenuate ischemic stroke in mice.^63,64 We found that compared to the scramble control (TAT-SC), TAT-M2 effectively inhibited the mitochondrial ROS production and Ca²⁺ overload induced by OGD (Figure 7A–D) or TSP1 treatment (Figure 7E–H) in CECs. Although TAT-M2 was shown to reduce the brain infarction after ischemic stroke in mice, but whether TAT-M2 produced other beneficial effects was undetermined.^63,64 We found that besides attenuating brain injury, TAT-M2 prevented the compromise of neurological functions 24 h after MCAO (Figure 7I–K). Also, TAT-M2 effectively inhibited Evans blue leakage (Figure 7I and L), suggesting that BBB integrity was preserved by TAT-M2.

Figure 7.

TRPM2 inhibition preserves BBB integrity in vivo. (A–D) Rh123 imaging and Fura-2 Ca²⁺ imaging after OGD. (A) Representative Rh123 images before and 15 min after OGD. (B) Quantification of changes of Rh123 fluorescence 15 min after OGD (n = 20–30/group). (C) Representative real-time Fura-2 Ca²⁺ imaging traces during OGD from 10 CECs. (D) Quantification of Fura-2 fluorescence changes 30 min after OGD (n = 20–30/group). (E–H) Rh123 imaging and Fura-2 Ca²⁺ imaging after TSP1 treatment. (E) Representative Rh123 images before and 10 min after TSP1 treatment. (F) Quantification of changes of Rh123 fluorescence 15 min after TSP1 treatment (n = 20–30/group). (G) Representative real-time Fura-2 Ca²⁺ imaging traces during TSP1 treatment from 10 CECs. (H) Quantification of Fura-2 fluorescence changes 20 min after TSP1 treatment (n = 20–30/group). (I) Evans blue assay and TTC staining of brain slices (1 mm). (J, K) Mean infarct volume and average ND score after MCAO from TAT-SC (n = 9) and TAT-M2 mice (n = 8). (L) Quantification of Evans blue assay after MCAO from WT (n = 7) and eM2KO mice (n = 6) (unpaired t-test with Welch’s correction).

In conclusion, our results suggest that the inhibition of TRPM2 is a promising therapeutic strategy in mitigating BBB degradation during ischemic stroke.

4. Discussion

The primary goal of therapies for ischemic stroke is to protect affected neurons. However, the failure of current treatments to directly inhibit neuronal death for mitigating ischemic brain injury is shifting researchers’ attention to non-neuronal cells.⁶ The NVU is a relatively new concept that emphasizes the importance of maintaining the homeostasis of the local microenvironment in the brain.^8,14 Endothelial cells compose the core component of the NVU and BBB, and endothelial hyperpermeability is a hallmark of brain damage during the early stages of ischemic stroke. This endothelial hyperpermeability drives the initial pathological changes of ischemic stroke as well as contributes to neuronal death.⁶⁵ However, the molecular mechanisms underlying endothelial dysfunction remain obscure.¹⁴

Global TRPM2 knockout protects mice against ischemic stroke, but the underlying mechanisms are still unclear.⁶⁶ Wild-type mice transplanted with bone marrow from Trpm2 knockout mice exhibited reduced brain injury after MCAO,²⁵ implying an important role of TRPM2 in core processes of ischemic stroke, including immune cell invasion and activation. Similarly, our previously published work showed that specific knockout of TRPM2 in myeloid lineage cells using CD11b-cre attenuates atherosclerosis in mice by inhibiting macrophage infiltration and activation.³⁹ Moreover, we recently found that neuronal TRPM2 aggravates brain injury by enhancing glutamate excitotoxicity.⁴⁴ Recently, methamphetamine and HIV-Tat protein were found to synergistically induce oxidative stress in CECs.⁶⁷ In this study, we demonstrated that selective deletion of Trpm2 in endothelial cells also effectively protects mice against ischemic stroke. This is the first study to report that TRPM2 in CECs plays a key role in ischemic stroke. Our results suggest that targeting TRPM2 could produce a comprehensive protective effect against ischemic stroke.

The role of TRPM2 in causing endothelial hyperpermeability has only been shown in lung-derived endothelial cells in vitro in response to H₂O₂ stimulation.²⁴ However, H₂O₂ treatment at a high concentration (300 µM) is not an ideal simulation of oxidative stress during in vivo ischemia.²⁴ In our study, we utilized OGD, which better mimics ischemic conditions.³⁷ We also used CECs instead of pulmonary endothelial cells, the latter of which are prone to hyperpermeability due to their loose tight junction, abundant fenestrations, and frequent pinocytotic activity.⁷

Phosphorylation of NOS3 at S1177 greatly enhances the activity of NOS3, and transgenic mice expressing the NOS3 S1177D, a phosphomimetic mutant, have been recently found to have robust NO production in the brain, which might protect the brain against oxidative stress.⁶⁸ We found that Trpm2 deletion or TRPM2 inhibition resulted in a marked increase in the phosphorylation of NOS3 at S1177 in the brain after MCAO, or in cultured CECs exposed to OGD, which is consistent with the inhibited ROS production evaluated by Rh123 mitochondrial imaging. These results provide strong evidence that the inhibition or deletion of Trpm2 protects against oxidative stress in CECs during ischemic stroke.

CD36 was shown to be critical for the Ca²⁺ influx induced by H₂O₂ in lung microvascular endothelial cells.⁶⁹ Endothelial CD36 promoted neutrophil activation after ischemic stroke and exacerbated brain damage.⁷⁰ We found that in isolated CECs, CD36 signaling cascades in CECs were activated by OGD to a similar degree compared to those activated by CD36 ligand TSP1. Recent single-cell RNA sequencing work identifies a distinct but abundant endothelial cell population with high CD36 expression.¹⁶ Our work suggests that CD36^high endothelial population may be prone to ischemic damage during stroke. Future fluorescence-activated cell sorting (FACS) work using CD36 should lead to better understanding of the role of CD36^low and CD36^high endothelial cells in different physiological and pathological conditions.

Previously, another CD36 ligand Aβ has been shown to induce TRPM2 activation in CECs, which caused neurovascular dysfunction in a Alzheimer’s disease mouse model,²⁶ albeit the underlying mechanism of Aβ-induced TRPM2 activation is not clear. The most intriguing discovery in our study is the activation of TRPM2 by CD36. In conjunction, activation of CD36 by OGD and TSP1 also requires TRPM2-mediated Ca²⁺ influx, suggesting the presence of a vicious TRPM2–CD36 cycle that enhances endothelial damage during ischemic stroke. This novel mechanism could also explain the Aβ-induced vascular damage in the development of Alzheimer’s disease.

TSP1 is a secreted glycoprotein produced by many cell types including endothelial cells. TSP1 is known for its inhibition of VEGF signaling by activating CD36 in endothelial cells,⁷¹ which causes inflammatory responses and induces apoptosis.⁵⁷ However, the underlying mechanisms regulating TSP1–CD36 signaling remain unclear. We found that Trpm2 deletion inhibited TSP1-induced CEC dysfunction and hyperpermeability. Also, TSP1 activates TRPM2 in a CD36-dependent manner. These results indicate that the detrimental effects of TSP1 on CECs are dependent on TRPM2 activation.

Another member of the TRPM family, TRPM4, is also crucial in facilitating ischemic brain injury. Recently, TRPM4 was shown to be associated with the NMDA receptor by enhancing its excitotoxicity in neurons.⁴⁶ Moreover, in CECs, TRPM4 associates with sulfonylurea receptor 1 (Sur1), which promotes BBB degradation and increases brain injury during ischemic stroke.^72–74 The activation of TRPM4 requires a marked increase of intracellular Ca²⁺,⁷⁵ and TRPM2 is highly permeable to Ca²⁺. We found that TRPM2 knockout could produce a similar protective effect against ischemic stroke as TRPM4 inhibition, which suggests that TRPM2-mediated Ca²⁺ signaling might be important for the activation of TRPM4.

One of the limitations of our study is using the young adult mice for MCAO model. As ischemic stroke is prevalent in elderly populations, our future studies will use aged mice and with comorbidities to better evaluate the therapeutic potential of targeting TRPM2 for ischemic stroke treatment.

In conclusion, we reveal that endothelial cell–specific TRPM2 knockout protects mice against ischemic stroke as evidenced by the reduced infarct volume and decreased plasma extravasation in the brain of mice subjected to MCAO. We discovered the mechanism by which deletion of Trpm2 in endothelial cells protects mice against ischemic stroke is through inhibiting oxidative stress, preserving tight junctions between CECs, and reducing immune cell invasion in the brain. At the cellular level, Trpm2 deletion protects CECs against oxidative stress and Ca²⁺ overload and thereby preventing endothelial hyperpermeability induced by OGD or TSP1. At the molecular level, we identified a novel mechanism that TRPM2 and CD36 are inter-dependently activated when CECs are exposed to OGD or TSP1. Our results establish that targeting endothelial TRPM2 is a promising strategy in mitigating ischemic brain injury.

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Authors’ contributions

L.Y. and P.Z. conceived and designed the research. P.Z. performed most in vitro experiments. C.X.L did some in vitro experiments. E.R.J. helped in and gave advice on flow cytometry analysis. Z.Y. and J.F. performed the in vivo experiments. B.M. generated TRPM2 floxp mice and provided inputs to discussion. P.Z. and L.Y. wrote the manuscript. All authors commented on the manuscript.

Funding

This work was partially supported by the National Institute of Health (R01HL147350 and R01NS131661) and American Heart Association (19TPA34890022) to L.Y.

Data availability

All data reported in this paper will be shared by the corresponding author upon request.