On June 25, 2024, the AHA Journals will be launching a new website design. During the launch process, there may be intermittent outages, and some features (alert sign-ups, article/issue purchases, account customizations/activations, and comment submissions) may be unavailable. This message will be removed when the launch process is complete. Thank you for your patience and we hope that you enjoy the new site!

Skip main navigation

LOX-1 and MMP-9 Inhibition Attenuates the Detrimental Effects of Delayed rt-PA Therapy and Improves Outcomes After Acute Ischemic Stroke

Originally publishedhttps://doi.org/10.1161/CIRCRESAHA.123.323371Circulation Research. 2024;134:954–969

Abstract

BACKGROUND:

Acute ischemic stroke triggers endothelial activation that disrupts vascular integrity and increases hemorrhagic transformation leading to worsened stroke outcomes. rt-PA (recombinant tissue-type plasminogen activator) is an effective treatment; however, its use is limited due to a restricted time window and hemorrhagic transformation risk, which in part may involve activation of MMPs (matrix metalloproteinases) mediated through LOX-1 (lectin-like oxLDL [oxidized low-density lipoprotein] receptor 1). This study’s overall aim was to evaluate the therapeutic potential of novel MMP-9 (matrix metalloproteinase 9) ± LOX-1 inhibitors in combination with rt-PA to improve stroke outcomes.

METHODS:

A rat thromboembolic stroke model was utilized to investigate the impact of rt-PA delivered 4 hours poststroke onset as well as selective MMP-9 (JNJ0966) ±LOX-1 (BI-0115) inhibitors given before rt-PA administration. Infarct size, perfusion, and hemorrhagic transformation were evaluated by 9.4-T magnetic resonance imaging, vascular and parenchymal MMP-9 activity via zymography, and neurological function was assessed using sensorimotor function testing. Human brain microvascular endothelial cells were exposed to hypoxia plus glucose deprivation/reperfusion (hypoxia plus glucose deprivation 3 hours/R 24 hours) and treated with ±tPA and ±MMP-9 ±LOX-1 inhibitors. Barrier function was assessed via transendothelial electrical resistance, MMP-9 activity was determined with zymography, and LOX-1 and barrier gene expression/levels were measured using qRT-PCR (quantitative reverse transcription PCR) and Western blot.

RESULTS:

Stroke and subsequent rt-PA treatment increased edema, hemorrhage, MMP-9 activity, LOX-1 expression, and worsened neurological outcomes. LOX-1 inhibition improved neurological function, reduced edema, and improved endothelial barrier integrity. Elevated MMP-9 activity correlated with increased edema, infarct volume, and decreased neurological function. MMP-9 inhibition reduced MMP-9 activity and LOX-1 expression. In human brain microvascular endothelial cells, LOX-1/MMP-9 inhibition differentially attenuated MMP-9 levels, inflammation, and activation following hypoxia plus glucose deprivation/R.

CONCLUSIONS:

Our findings indicate that LOX-1 inhibition and ± MMP-9 inhibition attenuate negative aspects of ischemic stroke with rt-PA therapy, thus resulting in improved neurological function. While no synergistic effect was observed with simultaneous LOX-1 and MMP-9 inhibition, a distinct interaction is evident.

Novelty and Significance

What Is Known?

  • Acute ischemic stroke remains a global burden leading to significant mortality and morbidity rates to which there are only 2 currently FDA-approved therapies, including endovascular thrombectomy and administration of rt-PA (recombinant tissue-type plasminogen activator). However, with these therapies remains an increased risk of hemorrhagic transformation leading to significant potential for worsened outcomes.

  • The endothelium plays a critical role in maintaining the homeostatic environment of the blood-brain barrier (BBB) and when perturbed during acute ischemic stroke can contribute significantly to the development and progression of stroke pathogenesis.

  • LOX-1 (lectin-like oxidized low-density lipoprotein receptor) is a key contributor to the pathogenesis of vascular diseases such as atherosclerosis. Moreover, LOX-1 has been linked to MMP-9 (matrix metalloproteinase 9), an enzyme strongly associated with cerebrovascular pathogenesis during acute ischemic stroke.

What New Information Does This Article Contribute?

  • We characterized the effects of acute ischemic injury, rt-PA administration, and LOX-1/MMP-9 inhibition on stroke outcomes using an in vivo as well as in vitro experimental stroke model.

  • We, in part, identified the impact of rt-PA on potential ischemic injury targets LOX-1 and MMP-9, as well as their role in the progression of acute ischemic stroke (AIS) at the functional, neurological, and molecular levels.

  • Using a thromboembolic stroke model, we characterized the spatiotemporal impact of ischemic stroke on MMP-9 activity within both the ipsilateral and contralateral hemispheric microvasculature and parenchymal tissues.

  • Additionally, we identified a spatiotemporal dysregulation of the cerebrovascular and parenchymal tissue microenvironment which was differentially altered by rt-PA in the presence and absence of novel pharmacological LOX-1 and MMP-9 inhibitors.

We demonstrate links between LOX-1 and MMP-9 as contributors that dysregulate barrier integrity, increase inflammation, and lead to worsened neurological function. We also highlight the detrimental effects of ischemic injury and rt-PA as well as the beneficial effects of selective LOX-1 and MMP-9 inhibitors at the level of the rodent cerebrovasculature and human brain microvascular endothelium. We propose that LOX-1 signaling leading to increased MMP-9 activity and neuroinflammation plays a critical role in worsened stroke pathogenesis.

In This Issue, see p 951

Meet the First Author, see p 952

Ischemic stroke affects over 9 million people each year worldwide, resulting in disability and death in 30% of the cases.1 Acute ischemic stroke (AIS) can trigger a complex and multimodal response at the level of the cerebrovasculature resulting in endothelial activation that, in part, disrupts vascular integrity setting off a cascade of secondary injuries that include barrier integrity loss, inflammation, edema, hemorrhagic risk, infarct expansion, and worsened stroke outcomes.2–5 Effective AIS therapies exist, such as thrombolysis and endovascular thrombectomy, which have improved stroke survivors’ quality of life.6,7 rt-PA (recombinant tissue-type plasminogen activator) remains the only FDA-approved drug for AIS therapy, however, due to its low efficacy, rt-PA administration is restricted to a therapeutic window of <4.5 hours after stroke onset and has been shown to elicit an increased risk of hemorrhagic transformation and can lead to considerable morbidity.6,8,9 It has also been recently recommended by the European Stroke Organization that patients with stroke onset between 4.5 and 9 hours in duration without CT or magnetic resonance imaging should not receive intravenous thrombolysis.10 However, for patients presenting within this same time frame with detailed mismatch imaging and exclusion for mechanical thrombectomy, the organization recommended intravenous thrombolysis accepting that usage of this therapy is an evolving area of investigation and further analysis is warranted.10

One of the underlying causes of increased hemorrhagic transformation risk appears to be due to the elevated levels of MMP-9 (matrix metalloproteinase 9),11 which is directly activated by rt-PA.12,13 Intriguingly, acute hemorrhagic transformation <18 to 24 hours following stroke onset has been postulated to be in part mediated by leukocyte-derived MMP-9 and brain-derived MMP-2 (matrix metalloproteinase 2).14 However, the contributions of the cerebrovasculature to increased blood concentrations of MMP-9 and worsened outcomes during the acute phase of AIS clinically remain to be elucidated.

Several processes can induce an increase of MMP-9 expression, one being through the signaling pathway of the LOX-1 (lectin-like oxLDL [oxidized low-density lipoprotein] receptor 1), a receptor that is activated during hypoxia15 and has been well characterized in other cardiovascular pathologies.16–19 In previous studies, we revealed a significant increase in LOX-1 levels 6 hours after ischemic stroke in hypertensive male and female animals.20,21 LOX-1 was first characterized and cloned in vascular endothelial cells22 and shown to induce endothelial cell dysfunction and activation. The connection between LOX-1 activation and MMP-9 production was established nearly a decade later in primary human aortic endothelial cells, which demonstrated that incubation with oxLDL induced an increase in levels of LOX-1, reactive oxygen species, NF-κB, and MMP-9 secretion.23

Although the LOX-1 receptor is capable of binding oxLDL, there are a multitude of other substrates that share few structural similarities to oxLDL such as C reactive protein, fibronectin, and aged/apoptotic cells all of which are capable of binding to this scavenger receptor.24 As such LOX-1 has been investigated in the context of vascular pathology such as atherosclerosis,25–28 diabetes type II,29 and in stroke30,31; therefore, targeting LOX-1 may be beneficial in mitigating the pathogenesis of AIS, even in the absence of elevated oxLDL levels. Moreover, at the level of the cerebrovascular endothelium, LOX-1 signaling in part increases MMP-9 activity and decreases tight junction protein expression.32 A significant portion of the endothelium’s regulatory role in maintaining blood-brain barrier (BBB) homeostasis is attributed to specific proteins such as claudin-5,33 occludin,34 and ICAM-1 (intercellular adhesion molecule 1),35,36 which comprise critical aspects of endothelial cell tight junction function and extravasation. This further suggests that LOX-1 may be a potential therapeutic target to in part attenuate brain endothelial dyshomeostasis during AIS progression.

Studies have concomitantly investigated the effect of late rt-PA treatment and the involvement of LOX-1 and MMP-9 detrimental outcomes on BBB integrity.37–39 Thus, it is feasible to hypothesize that inhibition of MMP-9 ± LOX-1 signaling in combination with rt-PA therapy could be a novel therapeutic approach to attenuate the increased risk of hemorrhagic transformation that accompanies rt-PA administration. Therefore, we hypothesized that LOX-1 ± MMP-9 inhibition would attenuate an ischemia/rt-PA-induced increase in MMP-9 activity, cerebrovascular permeability, and oxidative stress, which would result in improved neuroprotection and positive outcomes following ischemic stroke. In this study, we (1) determined the spatiotemporal dependent alteration in MMP-9 activation and LOX-1 levels within the cerebrovasculature and parenchymal tissues following stroke, (2) characterized the potential detrimental impact of rt-PA on the cerebrovasculature and functional outcomes following stroke as well as assessed the beneficial effects LOX-1 and MMP-9 inhibition, both separately and when administered in combination, (3) directly assessed endothelial cell barrier integrity, and (4) examined potential underlying mechanisms of cerebrovascular endothelial cell–derived MMP-9, integrity, and inflammation in response to ischemic injury, rt-PA, and combination therapy of LOX-1 and MMP-9 inhibition.

METHODS

Data Availability

The data supporting findings are available from the corresponding authors upon reasonable request. Detailed methods, study design, and statistical analysis using the in vivo and in vitro stroke models are available in Supplemental Material. In brief, thromboembolic stroke was performed in 12-week-old male Wistar rats (Janvier, France) as previously described.40

Although females were not included in this study, we recognize the importance of studying both sexes, and future investigations are aimed at including male and female stroke models to identify sex differences utilizing intense imaging, neurological tests, and molecular assays. All procedures and animal experiments were performed in compliance with the European Community Council Directive (2010/63/EU) for Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes guidelines. The ethical permit (Animal Inspectorate License No. 5.8.18-10593/2020) was approved by the Malmö-Lund Institutional Ethics Committee under the Swedish National Department of Agriculture. Results generated are reported in compliance with the Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines. Key elements of the study design are included in Supplemental Material.

RESULTS

Spatiotemporal Alterations in MMP-9 Activity Correlate With LOX-1 Expression Following Thromboembolic Stroke

MMP-9 activity via zymography at 3, 6, and 24 hours following the onset of thromboembolic stroke is represented in Figure 1A. MMP-9 activity in isolated brain vessels (Figure 1B and 1C) and parenchymal tissues (Figure 1D and 1E) from contralateral and ipsilateral hemispheres was increased at 6 hours post stroke onset, this response was specific to the ipsilateral hemisphere. Because LOX-1 has been well investigated in the context of vascular diseases such as atherosclerosis,26 hypertension,19 and stroke,20 we assessed the correlation between LOX-1 levels and increased MMP-9 activity. Although we did not observe changes in LOX-1 levels within the cerebral vasculature and parenchymal tissues following thromboembolic stroke (Figure S1A through S1F), we did note that within individual rats, there was a significant correlation between increased LOX-1 expression and MMP-9 activity within the cerebrovasculature in the ipsilateral hemisphere of injury (Figure S1G).

Figure 1.

Figure 1. Spatiotemporal brain microvascular and parenchymal MMP-9 (matrix metalloproteinase 9) activity following thromboembolic stroke. A, Representative gelatin zymography illustrating the band migration and enzymatic activity for MMP-9 (92 kDa) in isolated brain microvascular tissue from the ipsilateral and contralateral hemispheres following either sham or thromboembolic stroke at 3, 6, and 24 hours. Bar graphs depict MMP-9 activity within isolated microvessels (B and C) and parenchymal tissue (D and E) from the ipsilateral (B through D) and contralateral (C through E) hemispheres relative to injury by inverse densiometric analysis and expressed as arbitrary units (AU)±SD. n=2 to 6 animals (data are mean±SD). D, Kruskal-Wallis test used.

Combination of MMP-9 and LOX-1 Inhibition With Delayed rt-PA Therapy Differentially Attenuated Vascular MMP-9 Activity, Reduced Hemorrhagic Transformation, Decreased Infarct Size, and Improved Neurological Function

The utilization of rt-PA in the presence of selective MMP-9 and LOX-1 inhibition treatment or combination was examined by evaluating infarct volume, edema percentage, hemorrhagic incidence, and perfusion using magnetic resonance imaging. Representative magnetic resonance imagings from all treated groups are illustrated in Figure 2A. We did not observe an increase in infarct volume with rt-PA given 4 hours post-occlusion compared with saline; however, when combining rt-PA treatment with BI-0115 (LOX-1 inhibitor), there was a decrease in the formation of large infarct lesions (Figure 2B). In our experimental thromboembolic model, delayed rt-PA (4 hours after occlusion) increased the risk of edema and hemorrhagic transformation percentage at 24 hours poststroke induction (Figure 2C and 2D). This edema formation was attenuated by the LOX-1 inhibitor, BI-0115 (Figure 2C). Moreover, while hemorrhages were present in some animals treated with the LOX-1 (BI-0115) and MMP-9 (JNJ0966) inhibitors alone or in combination, the number and size of the hemorrhages were relatively smaller in comparison to the rt-PA-treated animals (Figure 2D). Perfusion within the ipsilateral hemisphere was restored in all animals 24 hours after stroke onset, except for animals treated with rt-PA and MMP-9 inhibition (Figure 2E). In terms of neurological function, we observed that following stroke, animals treated with saline, rt-PA, and rt-PA+JNJ0966 (MMP-9 inhibitor) had reduced functionality in their contralateral forepaw at 24 hours (Figure 2F). Animals treated with BI-0115 either alone or in combination with JNJ0966 plus rt-PA exhibited forepaw utilization similar to the sham-operated animals (Figure 2F). Comparable results were also observed in the broader sensorimotor test of neurological function (28-point neurological score). While delayed rt-PA therapy appeared to have further detrimental effects on stroke outcomes compared with saline-treated animals, LOX-1 inhibition significantly improved neurological function in animals treated with rt-PA (Figure 2G). Together these data suggest that targeting LOX-1 following a thromboembolic stroke is in part acting at the level of the BBB resulting in neuroprotection. We next assessed vascular-derived MMP-9 activity (Figure 2H) and LOX-1 protein expression (Figure 2I) within the ipsilateral hemisphere. In animals treated with saline at 24 hours poststroke, we observed no increase in MMP-9 activity within the vasculature both within the ipsilateral (Figure 2H) and contralateral (Figure S2A) hemispheres relative to sham. However, the administration of delayed rt-PA following the induction of stroke increased vascular-derived MMP-9 activity within the ipsilateral hemisphere compared with sham and was attenuated by JNJ0966 (Figure 2H). Concomitantly, rt-PA alone or conjugated with MMP-9 ± LOX-1 inhibitors decreased vascular-derived MMP-9 activity in the contralateral hemisphere compared with sham (Figure S2A). This suggests that administration of rt-PA at 4h poststroke onset spatially increased MMP-9 activity within the ipsilateral hemisphere of injury at 24-hour poststroke onset, which clinically correlates to an increased risk of hemorrhagic transformation as observed previously. Moreover, the selective MMP-9 inhibitor, JNJ0966, reduced the rt-PA-mediated increase in MMP-9 activity suggesting a potential protective effect on BBB integrity following stroke. In terms of LOX-1, the administration of rt-PA alone following stroke increased LOX-1 levels in the cerebrovasculature within the ipsilateral hemisphere and treatment with the MMP-9 or LOX-1 inhibitors attenuated this response (Figure 2I). Unlike that observed on the ipsilateral side, there was no effect of MMP-9 or LOX-1 inhibition on the cerebrovascular contralateral side (Figure S2B). Together, these data further support the positive correlation analysis of LOX-1 protein and MMP-9 activity levels within the vasculature, suggesting a detrimental link between LOX-1 and MMP-9 following AIS.

Figure 2.

Figure 2. Impact of rt-PA (recombinant tissue-type plasminogen activator)±LOX-1 (lectin-like oxidized low-density lipoprotein receptor 1) ± MMP-9 (matrix metalloproteinase 9) inhibition following thromboembolic stroke on infarct, edema, hemorrhage, cerebral blood flow, neurological outcome, MMP-9 activity, and LOX-1 expression. A, Representative T2- and T2*-weighted magnetic resonance imaging (MRI) as well as pCASL coronal images of rat brains at 24-hour reperfusion and postischemic injury in groups treated with ±rt-PA (3 mg/kg) in combination with JNJ0966 (10 mg/kg), BI-0115 (10 mg/kg), or JNJ+ BI-0115. B, Infarct volume as measured by MRI analysis and volume expressed as millimeter cube±SD. n=7 to 10 animals. C, Edema as measured by MRI analysis and expressed as the percentage change from sham±SD. n=8 to 10 animals. D, Hemorrhage as measured by MRI analysis and volume expressed as millimeters3±SD.n=8 to 9 animals. E, Cerebral blood flow (CBF) as measured by MRI analysis and expressed mL/100 g per minute±SD. n=6 to 8 animals. F, Bar graph depicting the percentage of injured paw usage measured before (baseline) and at 24 hours following treatment. n=7 to 12 animals. G, Bar graph depicts neurological score measured by blinded analysis and expressed as a scale between 0 and 28 with 0 representing the most severe and 28 representing the least severe. n=8 to 17 animals. H, MMP-9 activity within isolated microvessels from the ipsilateral hemisphere at 24-hour reperfusion and postischemic injury relative to injury by inverse densiometric analysis and expressed as arbitrary units (AU)±SD. n=6 to 9 animals. I, Densiometric analysis of LOX-1 protein levels expressed as AU normalized to β-actin±SD. n=5 to 9 animals. For data in B, D, G, I, a Kruskal-Wallis test was used. BI indicates BI-0115; JNJ, JNJ0966; and PCASL, pseudo-continuous arterial spin labeling.

Temporal Effect of Ischemic-Like Injury on Barrier Integrity and MMP-9 Activity in Human Brain Microvascular Endothelial Cells

We assessed functional barrier integrity via transendothelial electrical resistance over the course of 24 hours in human brain microvascular endothelial cells (HBMECs) following either normoxia or glucose deprivation (Figure 3). Following glucose deprivation, we observed an immediate decrease in both HBMEC impedance and resistance beginning at 10 minutes compared with normoxia with no difference in capacitance, suggesting a functional decrease in endothelial barrier integrity and function due to an increase in paracellular permeability (Figure 3B through 3D). We therefore next examined mRNA levels of various markers of inflammation and barrier integrity following 3 hours of hypoxia plus glucose deprivation (HGD) and during 12- and 24-hour reperfusion (HGD/R; Figure 3F through 3K). We concomitantly observed an increase in MMP-9 and IL-1β as well as a decrease in TIMP-1 (tissue inhibitors of metalloproteinases 1) mRNA levels in HBMECs following 3-hour HGD exposure and at 24-hour HGD/R (Figure 3F through 3H). Counter to our hypothesis, there was an initial increase in CLDN5 mRNA at 3 hours of HGD followed by a decrease at 24 hours of simulated reperfusion (Figure 3I). In contrast, there was an increase in occludin mRNA at 24 hours of simulated reperfusion (Figure 3J), which may suggest a preferential switch to occludin rather than claudin-5 expression following simulated ischemia-reperfusion injury. We observed a decrease in ICAM-1 mRNA at 3-hour HGD exposure with no relative change at 12 or 24 hours of simulated reperfusion (Figure 3K). Zymography of conditioned media revealed 2 distinct bands at approximately 93 and 65 kDa, corresponding to MMP-9 and MMP-2, respectively (Figure 3L and 3M). In concordance with our observations in saline-treated rats (Figure 2F), there was no difference in MMP-9 following in vitro HGD/R injury (Figure 3L). Similarly, extracellular HBMEC-derived MMP-2 activity was not different following HGD/R. (Figure 3M). We also assessed cell viability and found a decrease in HBMEC viability following HGD/R (Figure 3N). These data suggest that at the level of the human cerebrovascular endothelium, there is a temporal effect of ischemia-reperfusion on mechanisms to increase inflammation and decrease endothelial barrier integrity, potentially via an MMP-9 mechanism leading to cell death.

Figure 3.

Figure 3. Temporal effect of in vitro ischemia/reperfusion injury on human brain microvascular endothelial cell (HBMEC) barrier function, inflammatory and barrier markers, MMP-9 (matrix metalloproteinase 9)/MMP-2 (matrix metalloproteinase 2) activity, and cell viability. A, HBMEC treatment timeline of transendothelial electrical resistance (TEER) with or without glucose deprivation, a component of in vitro ischemic injury, for 24 hours. B through D, Graphs and representative tracings of impedance (B), resistance (C), and capacitance (D) are shown as the change relative to baseline, which was established 1 hour before glucose deprivation. n=4 independent samples. E, Treatment timeline of in vitro hypoxia plus glucose deprivation/reperfusion (HGD/R) injury on HBMECs exposed to either normoxia or HGD for 3 hours followed by either immediate collection or collection at 12 and 24 hours after reperfusion. F through K, qRT-PCR-mediated quantitation of MMP-9, TIMP-1 (tissue inhibitors of metalloproteinases 1), IL-1β, claudin-5, occludin, and ICAM-1 (intercellular adhesion molecule 1) mRNA expression normalized to the housekeeping gene (GAPDH) and expressed as 2−∆∆Ct. Samples were run in duplicate (data are mean±SD). n=4 to 9 independent samples. L and M, Representative gelatin zymography gels illustrating the band migration and enzymatic activity for 2 distinct matrix metalloproteinase bands, MMP-9 (93 kDa) and MMP-2 (65 kDa), in conditioned media collected from HBMECs. Bar graphs depict extracellular MMP-9 (L) and MMP-2 (M) activity by inverse densiometric analysis and expressed as fold change ±SD. n=8 independent samples. N, Graph illustrates cell viability of HBMECs, represented as live cell count/total cell count and expressed as fold change±SD. n=3 independent samples. F and K, Mann-Whitney U test used. IL-1β indicates interleukin 1 beta; and qRT-PCR, quantitative reverse transcription PCR.

Endothelial Barrier Function Is Increased by Selective LOX-1 Inhibition

We next assessed the impact of LOX-1 and MMP-9 inhibition on HBMEC barrier function by measuring transendothelial electrical resistance over a time course of 24 hours (Figure 4A). Following administration of inhibitors, we observed an immediate decrease in both HBMEC impedance and resistance beginning at 20 minutes, which was sustained for 4 hours with no difference in capacitance (Figure 4B through 4D). Although we saw an initial decrease in barrier integrity during the acute phase, all groups returned to the same resistance and impedance levels as a vehicle within 8 hours; however, selective LOX-1 inhibition significantly improved barrier integrity as measured by increased resistance beginning at 16 hours (Figure 4C), suggesting that LOX-1 inhibition has the potential to improve endothelial barrier function. We hypothesize that a similar increase in barrier function was not observed following MMP-9 inhibition due to the notable low levels of MMP-9 activity previously observed in HBMECs under normoxia conditions. To interrogate potential mechanisms underlying these observations, we examined the impact of LOX-1 ± MMP-9 inhibition on mRNA levels of various markers of inflammation and barrier integrity following either normoxia or HGD/R (Figure 4E). Intriguingly, we did not observe an appreciable effect of selective LOX-1 ± MMP-9 inhibition on claudin-5, TIMP-1, or MMP-9 mRNA levels under normoxic conditions (Figure 4F through 4H). However, we did observe that MMP-9 inhibition alone increased claudin-5 and LOX-1 inhibition alone or combined led to decreased MMP-9 mRNA levels during HGD/R injury. When MMP-9 or MMP-9 plus LOX-1 inhibitors were given, we observed an attenuative effect on the restoration of claudin-5 mRNA levels mediated by MMP-9 inhibition alone. This finding is interesting and warrants further investigation however falls beyond the scope of this study. We assessed cell viability and observed that LOX-1 inhibition alone or in combination with MMP-9 inhibition attenuated HGD/R-mediated increases in cell death (Figure 4I). Together, these data suggest that selective LOX- 1 inhibition increases basal endothelial barrier integrity function and that MMP-9 ± LOX-1-inhibition exhibit differential effects on claudin-5 and MMP-9 expression as well as improve cell viability.

Figure 4.

Figure 4. Temporal effect of selective LOX-1 (lectin-like oxidized low-density lipoprotein receptor 1) ±MMP-9 (matrix metalloproteinase 9) inhibition on human brain microvascular endothelial cell (HBMEC) barrier function as well as claudin-5, TIMP-1 (tissue inhibitors of metalloproteinases 1), and MMP-9 mRNA, and cell viability following hypoxia plus glucose deprivation/reperfusion (HGD/R). A, Treatment timeline of in vitro barrier function measured as transendothelial electrical resistance (TEER) in HBMECs exposed to vehicle, MMP-9 inhibition (JNJ0966, 5 μM), LOX-1 (lectin-like oxidized low-density lipoprotein receptor 1) inhibition (BI-0115, 10 μM), or MMP-9 plus LOX-1 inhibition for 24 hours. Graphs and representative tracings of (B) impedance, (C) resistance, and (D) capacitance are shown as the change relative to baseline, which was established 1 hour before drug administration. n=4 independent samples. E, Treatment timeline of in vitro ischemia/reperfusion injury on HBMECs exposed to either normoxia or HGD/R and treated with either vehicle, MMP-9 inhibition (JNJ0966, 5 μM), LOX-1 inhibition (BI-0115, 10 μM), or MMP-9 plus LOX-1 inhibition for 24 hours. qRT-PCR mediated quantitation of (F) claudin-5, (G) TIMP-1, and (H) MMP-9 mRNA expression normalized to the housekeeping gene (GAPDH) and expressed as 2−∆∆Ct. Samples were run in duplicate (data are mean±SD). n=3 independent samples. I, Cell viability of HBMECs, represented as live cell count/total cell count and expressed as fold change±SD. n=3 independent samples. BI indicates BI-0115; JNJ, JNJ0966; and and qRT-PCR, quantitative reverse transcription PCR.

MMP-9 Plus LOX-1 Inhibition in Combination With rt-PA Elicits Differential Effects on HBMEC MMP-9/2 Activity As Well As MMP-9, Inflammation, Tight Junction, and Adhesion Molecule mRNA Expression

To address potential mechanisms promoting cerebrovascular permeability following an ischemic-like injury and in the context of rt-PA, we assessed MMP-9/2 activity and mRNA expression of multiple mediators in HBMECs following HGD 3 hours/R 24 hours in the presence or absence of MMP-9/LOX-1 inhibition (Figure 5A). We observed that rt-PA therapy did not alter HBMEC-derived MMP-9 activity at 24 hours after reperfusion (Figure 5B). However, there was a significant increase in MMP-2 activity at HGD/R with rt-PA compared with the vehicle (Figure 5C). Interestingly, rt-PA therapy alone or in combination with MMP-9/LOX-1 inhibition attenuated MMP-9 mRNA expression (Figure 5D). rt-PA alone did not alter TIMP-1 mRNA expression, however, when treated with rt-PA plus MMP-9/LOX-1 inhibition, TIMP-1 mRNA levels increased (Figure 5E). We next assessed HBMEC IL-1β mRNA expression and found that treatment with rt-PA significantly increased IL-1β mRNA levels, which was attenuated by MMP-9/LOX-1 inhibitor cotherapy (Figure 5F). Additionally, we measured levels of extracellular hydrogen peroxide (H2O2) to further assess potential mechanisms behind MMP-9 activation and observed that HGD/R increased levels of HBMEC-derived extracellular H2O2 when compared with normoxic controls; however, rt-PA alone or in combination with MMP-9/LOX-1 inhibition had no effect (Figure S3A and S3B). Moreover, we observed no difference in the levels of SOD1 mRNA under any exposure/treatment conditions (Figure S3C and S3D). Overall, these findings suggest that combination therapy of MMP-9/LOX-1 inhibition with rt-PA could promote protective effects on HBMEC-derived MMP-9 and inflammation following HGD/R injury. To assess potential underpinnings of endothelial integrity in the context of rt-PA and whether MMP-9/LOX-1 inhibition alters this response, we measured mRNA expression of tight junctions and adhesion molecule markers. We observed decreased CLDN5 mRNA expression following rt-PA therapy alone or in the presence of MMP-9/LOX-1 inhibition (Figure 5G). In contrast to CLDN5 mRNA expression, there was an increase in OCLN mRNA in HBMECs treated with the combination of MMP-9/LOX-1 inhibition plus rt-PA therapy compared with vehicle (Figure 5H) furthering our hypothesis that following HGD/R there might be a preferential switch within HBMECs toward occludin expression rather than claudin-5. This is supported by our observations that HGD/R decreased mRNA expression of zonula occludens 1 (TJP1; Figure S3E), which is critical for the stabilization of claudin-5 at the BBB (reviewed in study by Lochhead et al41). Moreover, we observed an increase in HBMEC ICAM-1 mRNA expression following rt-PA therapy compared with HGD/R alone (Figure 5I). In efforts to identify concomitant effects of HGD/R on mRNA in the presence or absence of rt-PA alone or in combination with MMP-9/LOX-1 inhibition, we performed Pearson correlation analysis (Figure 5J through 5R). Breakdowns of each correlation can be found in Table S1. Together, these data suggest that HGD/R mediates a potential decrease in endothelial barrier integrity via a decrease in CLDN5 and TJP1 mRNA. Furthermore, rt-PA therapy may exacerbate this barrier dysfunction by differentially altering CLDN5 and OCLN and increasing ICAM-1 mRNA levels, which is partially attenuated by the combination therapy of MMP-9/LOX-1 inhibition with rt-PA.

Figure 5.

Figure 5. Impact of hypoxia plus glucose deprivation/reperfusion (HGD/R) ±rt-PA (recombinant tissue-type plasminogen activator) ±LOX-1 (lectin-like oxidized low-density lipoprotein receptor 1) and MMP-9 (matrix metalloproteinase 9) inhibition on human brain microvascular endothelial cell (HBMEC) MMP-9/2 activity, MMP-9, inflammatory, and barrier markers. A, Treatment timeline. B and C, Representative gelatin zymography gels illustrating the band migration and enzymatic activity for 2 distinct matrix metalloproteinase bands, MMP-9 (93 kDa) and MMP-2 (65 kDa), in conditioned media collected from HBMECs. Bar graphs depict extracellular (B) MMP-9 and (C) MMP-2 activity by inverse densiometric analysis and expressed as fold change ±SD. n=8 independent samples. Also depicted, qRT-PCR (quantitative reverse transcription PCR) quantitation of (D) MMP-9, (E) TIMP-1, (F) IL-1β, (G) claudin-5, (H) occludin, and (I) ICAM-1 (intercellular adhesion molecule 1) mRNA normalized to the housekeeping gene (GAPDH) and expressed as 2−∆∆Ct. n=3 to 6 independent samples. J through L, Graphical representation of Pearson correlation matrices of HBMEC mRNA levels on a scale from −1 to 1 following HGD/R and either treated with (J) vehicle, (K) rt-PA, or (L) JNJ0966+BI-0115+rt-PA. n=4 to 6 independent samples. M through R, Graphical representation of Pearson correlation of extracellular HBMEC MMP-9 or MMP-2 activity against mRNA levels following HGD/R and either treated with (M and P) vehicle, (N and Q) rt-PA, or (O and R) JNJ0966+BI-0115+rt-PA. M through R, n=4 to 8 independent samples.

MMP-9 Activity Is Correlated With Worsened Stroke Outcomes

To further elucidate the detrimental role of MMP-9 in the pathogenesis of preclinical stroke outcomes such as edema, infarct volume, and neurological function, we performed correlative analyses of vascular MMP-9 activity with these outcomes across all treatment groups. The individual treatment groups can be found in the Table. Following correlative analysis, we determined that increased MMP-9 activity at 24 hours after thromboembolic stroke was significantly correlated with increased edema (Figure 6A), worsened neurological function (Figure 6B), and increased infarct volume (Figure 6C). These data suggest that targeting vascular-derived MMP-9 either by direct steric inhibition or potentially through the combination therapy of LOX-1 and MMP-9 inhibition could lead to improved stroke outcomes.

Table. Correlation of Cerebrovascular MMP-9 Activity, Edema, Neurological Function, and Infarct Volume Broken Down by Treatment Condition

Treatment conditionIndependent variableDependent variableSlope (95% CI)Y-intercept (95% CI)R squareF (DFn, DFd)P value (effect size of independent variable)
ShamMMP-9Edema1332 (−3545, 6210)−128 554 (−620 139, 363 032)0.12570.5753 (1, 4)0.4904
SalineMMP-9Edema315.8 (−1913, 2544)−20 151 (−252 024, 211 722)0.025850.1327 (1, 5)0.7305
rt-PAMMP-9Edema2057 (−13 452, 17 565)−200 589 (−1 847 556, 1 446 377)0.032780.1356 (1, 4)0.7314
JNJ+rt-PAMMP-9Edema364.7 (−4117, 4846)−27 919 (−496 113, 440 274)0.0052620.03703 (1, 7)0.8529
BI+rt-PAMMP-9Edema3239 (−14 986, 21 463)−298 202 (−2 215 616, 1 619 212)0.040070.2087 (1, 5)0.6669
JNJ+BI+rt-PAMMP-9Edema1758 (−1611, 5127)−169 263 (−526 384, 187 859)0.47902.759 (1, 3)0.1953
ShamMMP-9Neurological scoreN/AN/A1.000N/AN/A
SalineMMP-9Neurological score−1227 (−3875, 1420)33 166 (−19 107, 85 440)0.086471.041 (1, 11)0.3295
rt-PAMMP-9Neurological score−4352 (−8395, −308.8)95 085 (23 485, 166 685)0.39715.929 (1, 9)0.0377*
JNJ+rt-PAMMP-9Neurological score−199.7 (−5397, 4998)15 232 (−85 960, 116 424)0.00073220.007327 (1, 10)0.9335
BI+rt-PAMMP-9Neurological score−8065 (−40 547, 24 418)196 810 (−451 489, 845 109)0.046930.3447 (1, 7)0.5756
JNJ +BI +rt-PAMMP-9Neurological score−2776 (−9838, 4286)66 959 (−61 015, 194 933)0.34281.565 (1, 3)0.2996
ShamMMP-9Infarct volume, mm3N/AN/A1.000N/AN/A
SalineMMP-9Infarct volume, mm3159.0 (6.578, 311.5)−1241 (−14 652, 12 171)0.58987.190 (1, 5)0.0437*
rt-PAMMP-9Infarct volume, mm3192.0 (−148.4, 532.4)1001 (−25 348, 27 351)0.24101.905 (1, 6)0.2168
JNJ+rt-PAMMP-9Infarct volume, mm374.29 (−120.7, 269.3)6582 (−7886, 21 050)0.067210.7205 (1, 10)0.4158
BI+rt-PAMMP-9Infarct volume, mm3−63.12 (−1080, 954.0)40 964 (−50 847, 132 775)0.0030670.02154 (1, 7)0.8875
JNJ+BI+rt-PAMMP-9Infarct volume, mm392.04 (−41.75, 225.8)7728 (−9874, 25 330)0.61514.793 (1, 3)0.1163

BI indicates BI-0115; JNJ, JNJ0966; MMP-9, matrix metalloproteinase 9; and rt-PA, recombinant tissue-type plasminogen activator.

* Denotes a significant effect size of independent variable following linear regression.

Figure 6.

Figure 6. Correlation between cerebrovascular MMP-9 (matrix metalloproteinase 9) activity and edema, neurological function, and infarct volume. Graphs depicting MMP-9 activity (y axis) observed in brain microvessels isolated from the ipsilateral hemisphere of rats postischemic injury and at 24-hour reperfusion and the corresponding edema (A), neurological score (B), and infarct volume (mm3; C; x axis). A, n=39 animals. B, n=62 animals.C, n=51 animals.

DISCUSSION

In our study design, we evaluated whether pharmacological application of MMP-9 ± LOX-1 inhibition in combination with rt-PA treatment, using a clinically relevant thromboembolic model of stroke model, limits or mitigates brain damage development. Additionally, we evaluated the role of MMP-9 ± LOX-1 inhibition on barrier integrity in both our in vivo experimental stroke model as well as in an in vitro human brain endothelial cell ischemia/reperfusion model. For the first time, we demonstrated that LOX-1 inhibition alone or potentially combined with MMP-9 inhibition conjugated with rt-PA significantly improved stroke outcome. In brief, we observed that stroke followed by rt-PA treatment increased (1) hemorrhage and edema, (2) cerebrovascular MMP-9 activity and LOX-1 levels, and (3) markers of inflammation and endothelial activation. MMP-9 ± LOX-1 inhibition differentially attenuated these responses (Figure 7).

Figure 7.

Figure 7. Schematic summary of cerebrovascular pathology of stroke and rt-PA (recombinant tissue-type plasminogen activator) as well as protective effects of MMP-9 (matrix metalloproteinase 9) ± LOX-1 (lectin-like oxidized low-density lipoprotein receptor 1) inhibition on brain endothelial integrity and health. BBB indicates blood-brain barrier.

Cerebral edema and hemorrhagic transformation are significant risks for the administration of thrombolysis beyond the 4.5-hour therapeutic window and have been linked to vascular disruptors, MMP-9 and LOX-1. Clinically, rt-PA therapy can increase the risk of edema, BBB disruption, and hemorrhagic transformation by 6% to 8%,42–44 which can lead to worsened outcomes.45 Mechanistically, these increases are in part due to rt-PA’s off-target effects involving MMP-9 activation46–48 as well as increasing parenchymal free radical production, leukocyte extravasation, and microglia aggravation.49 The detrimental effects of rt-PA therapy depend highly on MMP-9 activation and breakdown of the BBB.11 In this study, we demonstrated that rt-PA treatment 4-hour poststroke onset induced a significant increase in MMP-9 activity in isolated cerebral vessels 24 hours after reperfusion (Figure 2H). In addition, the delayed treatment with rt-PA resulted in worsened neurological function 24 hours after stroke onset (Figure 2F and 2G). Similar results were previously observed in the same stroke model where delayed rt-PA therapy increased the risk of hemorrhagic transformation and increased the percentage of edema.40 This mimics clinical observations where delayed administration of rt-PA was provided to patients, thus indicating the relevance of this thrombolysis stroke model study.11,50–52

Due to the increase in MMP-9 activity at 6-hour postthromboembolic stroke onset (Figure 1B and 1D), we hypothesized that administration of the LOX-1 and MMP-9 inhibitors prior to this time point would have the most optimal effect on the safety of the treatment. Therefore, we administered the inhibitors 3.5 hours after stroke onset and 30 minutes before the rt-PA therapy. Administration of these selective inhibitors before rt-PA is advantageous as rt-PA treatment comes with an extensive list of contraindications53 delaying treatment in patients when they first arrive in the clinic. Treatment with either or both LOX-1/MMP-9 inhibitors may not require this exclusionary step, making it possible to treat patients on arrival before rt-PA therapy, and increasing translation to the clinic. Our observations of increased MMP-9 activity both within the parenchymal and isolated cerebrovascular tissues closely mimic the previously observed increase in homogenized brains of rats subjected to 1 hour of transient middle cerebral artery occlusion, which demonstrated a trend increase in MMP-9 activity beginning at 4 hours post-injury.54 Together suggesting the importance of ischemic model selection, as the resolved occlusion and addition of reperfusion appears to increase MMP-9 activity both within the ipsilateral cerebrovascular and parenchymal tissue at 6 hours post-onset, which was then decreased at 24 hours post-onset (Figure 1B through 1E).

The complex role of the endothelium in maintaining a functional barrier is challenged by the enzymatic activity of MMP-9, which has been considered clinically as a marker of ischemic stroke55 and shown to disrupt the cerebrovascular basal lamina to actively exacerbate barrier permeability. A study in a murine model of intracerebral hemorrhage reported that stimulated MMP-9 levels colocalized with cerebral microvessels,56 suggesting that injury-activated endothelium may play a significant role in the production of MMP-9. Similarly, in our thromboembolic stroke model, rt-PA administration following 24 hours of stroke increased cerebrovascular MMP-9 activity in rat brain microvasculature (Figure 2H). Additionally, rt-PA led to an increase in HBMEC-derived MMP-2 activity following HGD/R injury (Figure 5C). We acknowledge that cultured endothelial cells do not identically match conditions observed in vivo; however, this is the first study to demonstrate that HBMECs were exposed to ischemia-reperfusion injury in the presence of rt-PA and further investigation into the temporal impact of the response of MMP-9 to these perturbations is warranted to elucidate potential mechanisms underlying this differential response. We also acknowledge that the complex pathophysiological cascade shown to occur during an ischemic stroke cannot be precisely modeled in an in vitro setting (ie, lack of supplemental BBB components such as the CNS, mechanotransduction effect of blood flow, and peripheral immune cell responses). However, in vitro studies using human primary cells prompt the investigation of specific basic cellular and molecular mechanisms under conditions of HGD plus reperfusion, which is like what is observed during AIS.

At the level of the vasculature, LOX-1 has been shown to induce vascular smooth muscle and endothelial dysfunction leading to impaired nitric oxide–mediated vasodilation and apoptosis.57–63 The overlapping activation of NF-κB both in the vascular smooth muscle and endothelium following LOX-1 activation has been shown to induce a positive feedback loop resulting in increased ORL1 transcription and increased LOX-1 protein.64 This activation of NF-κB has also been linked to increased MMP-9 transcription,65,66 which has been highly implicated with the worsening of ischemic injury pathogenesis.67–70 Previous efforts to address the role of LOX-1 and its subsequent detrimental role in vascular pathology such as atherosclerosis25–28 and stroke30,31 support the potential beneficial effect of targeting LOX-1 to attenuate acute ischemic injury sequelae. Recently, it has been shown that deletion of LOX-1 had protective effects in stroke-prone spontaneously hypertensive rats, which based on miRNA biomarker analysis, was proposed to be acting to attenuate LOX-1-induced dysfunction at the level of the BBB following cerebral ischemia.30 It has been previously found that mice overexpressing endothelial LOX-1 had increased stroke volumes and worsened neurological function at 24 hours following transient middle cerebral occlusion and treatment by LOX-1 silencing RNA had the reverse effect on these outcomes.71 Together suggesting that targeting LOX-1 in the management and treatment of AIS may be a viable approach.

Based on the previously established connection between LOX-1 and MMP-9, we hypothesized that animals with an increased concentration of cerebrovascular LOX-1 protein would have increased MMP-9 activity following stroke which would lead to worsened outcomes. In this study, we observed from 17 individual rats subjected to thromboembolic stroke that the ipsilateral cerebrovasculature LOX-1 protein levels were positively correlated with MMP-9 activity (Figure S1G). This is the first time that LOX-1 and MMP-9 levels and activity have been correlated within the cerebrovasculature following acute cerebral ischemic injury. While there has been no such previous connection established between these proteins within the cerebrovasculature under similar experimental conditions, there remains sufficient literature linking the LOX-1 to MMP-9 at the transcriptional and activation levels.

In this study, overall, we observed that the detrimental effects of stroke and rt-PA treatment were differentially attenuated by LOX-1 and MMP-9 inhibition individually and in some instances had similar effects. LOX-1 inhibition in combination with rt-PA therapy reduced the infarct area as well as attenuated edema (Figure 2B and 2C). We also observed that LOX-1 inhibition in HBMECs improved barrier function (Figure 4B and 4C). These findings complement a previous study, which demonstrated that a deficiency or neutralization of LOX-1 reduced the infarct volume in a murine model of stroke at 24 hours post-injury onset following a 2-hour middle cerebral artery occlusion.72 Moreover, a separate study performed in rats on postnatal day 7 subjected to hypoxic-ischemic injury for 2 hours demonstrated that anti-LOX-1 antibody reduced edema as well as rescued tight junction proteins ZO-1 (zona occludins-1) and occludin at 48 and 72 hours post-injury.73 Additionally, in this study, we observed that targeting LOX-1 and MMP-9 separately and together reduced the number of large hemorrhagic events (Figure 2D). These results confer with previously established reports demonstrating that inhibition of MMP-9 attenuates the increased risk of hemorrhagic transformation.74 However, this is the first demonstration of the protective effects via LOX-1 inhibition conjugated with rt-PA on hemorrhagic transformation. This observed beneficial efficacy was also observed at the neurological level, which seems to be LOX-1 driven (Figure 3F and 3G). It has been previously demonstrated in a rat model of cerebral ischemia/reperfusion injury that attenuating MMP-9 activity during the acute phase results in improved neurological function.75 However, unlike the 2013 study by Zhao et al, we were unable to detect an improvement in neurological function as determined by the percent usage of injured forepaw in the rats treated with the MMP-9 inhibitor in conjunction with rt-PA (Figure 2F). In this model, stroke plus the addition of rt-PA had a negative impact on outcomes as time from onset increased, a phenomenon also seen in the clinic where the efficacy of rt-PA decreases with time. Here, we show that in a model where the effectiveness of rt-PA is zero in the outcomes measured, we still detect differential improvement when combined with LOX-1 inhibition. This improvement in neurological function may be in part due to the antiapoptotic effect previously observed by blocking LOX-1 in neonatal rats subjected to hypoxia ischemic injury,73 as well as the barrier enhancement and cell survival observations within HBMECs (Figure 4B, 4C, and 4I).

While the MMP-9 inhibitor had a more considerable influence on the MMP-9 activity than the LOX-1 inhibitor, the utilization of either LOX-1 alone or the combination of both may be enough to improve outcomes following stroke and rt-PA therapy. Intriguingly, we observed that not only did the selective MMP-9 inhibitor decrease MMP-9 activity, but it also decreased cerebrovascular LOX-1 protein levels 24 hours post-injury (Figure 2I). It has been previously demonstrated that LOX-1 activation can induce a positive feedback loop in which more LOX-1 protein is produced76 and in line with this previous report we report that the novel selective LOX-1 inhibitor reduced LOX-1 protein levels. However, contrary to what we expected, LOX-1 levels were not decreased in the cerebrovasculature of rats treated with the combination of both LOX-1 and MMP-9 inhibitors in addition to rt-PA which merits further investigation.

Based on our results in this study, we posit that LOX-1 inhibition alone increases HBMEC barrier function, and attenuates ischemia-reperfusion–mediated cell death and MMP-9 transcription. We also hypothesize that the combination of LOX-1 and MMP-9 inhibitors is in part working at the level of the endothelium to reduce focal inflammation (IL-1β), endothelial activation (ICAM-1), and rescue tight junction expression (occludin) within the HBMEC to mitigate ischemia/reperfusion-induced decreases in cerebrovascular barrier integrity. Here, we confirmed that MMP-9 is significantly associated with adverse outcomes such as increased edema, infarct volume, and decreased neurological score as well as that inhibition of LOX-1 alone or potentially in combination with MMP-9 inhibition may be beneficial to reduce stroke and rt-PA therapy–induced negative outcomes. These previous data in Supplemental Material demonstrate that targeting the vasculature can lead to improved stroke outcomes.74,77,78 BI-0115 is a selective LOX-1 inhibitor that prevents the binding of the LOX-1 receptor and therefore inhibits the signaling cascade and possibly the upregulation of MMP-9. In comparison, JNJ0966 is a direct MMP-9 inhibitor that prevents the activation of MMP-9 by binding allosterically to the protease. In our study, a nondiscernable additive or synergistic action between LOX-1 and MMP-9 was observed. These events may involve distinct mechanisms: (1) LOX-1 activation inducing increased MMP-9 mRNA expression and (2) LOX-1 blockade effectively inhibiting its activity but not necessarily blocking MMP-9 activity. This may suggest that these molecules operate at separate regulatory levels within endothelial cells, exerting distinct effects in a nonadditive or synergistic manner.

In conclusion, our findings provide the first evidence that a combination of MMP-9 ± LOX-1 inhibition attenuates aspects of cerebrovascular pathology following ischemic stroke with rt-PA therapy, thus resulting in improved neurological function. Additionally, at the level of the cerebrovasculature, our data suggest that MMP-9 ± LOX-1 inhibition attenuates MMP-9 activity and LOX-1 levels in vivo as well as endothelial markers of inflammation, activation, and barrier integrity loss following ischemic-like injury providing a potential mechanism that lays the groundwork for future cerebrovascular based therapeutic strategies for stroke treatment. The focus of this study is on potentially extending the window of opportunity for rt-PA therapy may have a significant impact on millions of patients who suffer from AIS. In addition, it also outlines upcoming directions to assess the efficacy of these potential novel therapeutics in models of ischemia-reperfusion injury in the absence of thrombolysis. This approach acknowledges the evolving nature of the clinical treatment, considering that some patients with an occlusion resolve their clots either without rt-PA or tenecteplase-mediated thrombolysis or are eligible to undergo mechanical thrombectomy.

ARTICLE INFORMATION

Acknowledgments

The authors are grateful to Lund University Bioimaging center for their assistance and providence of the magnetic resonance imaging facility. They also thank Dr Emmanuel Barbier at Grenoble Institute of Neurosciences for sharing the method for pseudocontinuous arterial spin labeling. BI-0115 was kindly provided by Boehringer Ingelheim via its open innovation platform opnMe, available at https://opnme.com.

Supplemental Material

Supplemental Methods

Major Resources Tables

Tables S1 and S2

Figures S1–S4

References 40,79–85

Nonstandard Abbreviations and Acronyms

AIS

acute ischemic stroke

BBB

blood-brain barrier

HBMEC

human brain microvascular endothelial cell

HGD

hypoxia plus glucose deprivation

HGD/R

hypoxia plus glucose deprivation/reperfusion

ICAM-1

intercellular adhesion molecule 1

LOX-1

lectin-like oxidized low-density lipoprotein receptor 1

MCA

middle cerebral artery

MMP-2

matrix metalloproteinase 2

MMP-9

matrix metalloproteinase 9

oxLDL

oxidized low-density lipoprotein

rt-PA

recombinant tissue-type plasminogen activator

TIMP1

tissue inhibitors of metalloproteinases 1

ZO-1

zona occludins-1

Disclosures None.

Footnotes

*K. Arkelius and T.S. Wendt contributed equally.

†R.J. Gonzales and S. Ansar contributed equally as senior authors.

For Sources of Funding and Disclosures, see page 967.

Supplemental Material is available at https://www.ahajournals.org/doi/suppl/10.1161/CIRCRESAHA.123.323371.

Correspondence to: Saema Ansar, PhD, Applied Neurovascular Research, Neurosurgery, Department of Clinical Sciences, Faculty of Medicine, Lund University, Lund, Sweden, Email
Rayna J. Gonzales, PhD, Department of Basic Medical Sciences, University of Arizona College of Medicine Phoenix, AZ, Email

REFERENCES

  • 1. Lindsay MP, Norrving B, Sacco RL, Brainin M, Hacke W, Martins S, Pandian J, Feigin V. World Stroke Organization (WSO): global stroke fact sheet 2019.Int J Stroke. 2019; 14:806–817. doi: 10.1177/1747493019881353CrossrefMedlineGoogle Scholar
  • 2. Kunz A, Iadecola C. Cerebral vascular dysregulation in the ischemic brain.Handb Clin Neurol. 2009; 92:283–305. doi: 10.1016/S0072-9752(08)01914-3CrossrefMedlineGoogle Scholar
  • 3. Hu X, De Silva TM, Chen J, Faraci FM. Cerebral vascular disease and neurovascular injury in ischemic stroke.Circ Res. 2017; 120:449–471. doi: 10.1161/CIRCRESAHA.116.308427LinkGoogle Scholar
  • 4. Nelson CW, Wei EP, Povlishock JT, Kontos HA, Moskowitz MA. Oxygen radicals in cerebral ischemia.Am J Physiol. 1992; 263:H1356–H1362. doi: 10.1152/ajpheart.1992.263.5.H1356CrossrefMedlineGoogle Scholar
  • 5. Abo-Ramadan U, Durukan A, Pitkonen M, Marinkovic I, Tatlisumak E, Pedrono E, Soinne L, Strbian D, Tatlisumak T. Post-ischemic leakiness of the blood-brain barrier: a quantitative and systematic assessment by Patlak plots.Exp Neurol. 2009; 219:328–333. doi: 10.1016/j.expneurol.2009.06.002CrossrefMedlineGoogle Scholar
  • 6. Hacke W, Kaste M, Bluhmki E, Brozman M, Davalos A, Guidetti D, Larrue V, Lees KR, Medeghri Z, Machnig T, et al; ECASS Investigators. Thrombolysis with alteplase 3 to 4.5 hours after acute ischemic stroke.N Engl J Med. 2008; 359:1317–1329. doi: 10.1056/NEJMoa0804656CrossrefMedlineGoogle Scholar
  • 7. Hacke W, Kaste M, Fieschi C, Toni D, Lesaffre E, von Kummer R, Boysen G, Bluhmki E, Hoxter G, Mahagne MH. Intravenous thrombolysis with recombinant tissue plasminogen activator for acute hemispheric stroke. The European Cooperative Acute Stroke Study (ECASS).JAMA. 1995; 274:1017–1025. PMID: 7563451CrossrefMedlineGoogle Scholar
  • 8. Bhatia R, Hill MD, Shobha N, Menon B, Bal S, Kochar P, Watson T, Goyal M, Demchuk AM. Low rates of acute recanalization with intravenous recombinant tissue plasminogen activator in ischemic stroke: real-world experience and a call for action.Stroke. 2010; 41:2254–2258. doi: 10.1161/STROKEAHA.110.592535LinkGoogle Scholar
  • 9. Lee KY, Han SW, Kim SH, Nam HS, Ahn SW, Kim DJ, Seo SH, Kim DI, Heo JH. Early recanalization after intravenous administration of recombinant tissue plasminogen activator as assessed by pre- and post-thrombolytic angiography in acute ischemic stroke patients.Stroke. 2007; 38:192–193. doi: 10.1161/01.STR.0000251788.03914.00LinkGoogle Scholar
  • 10. Berge E, Whiteley W, Audebert H, De Marchis GM, Fonseca AC, Padiglioni C, de la Ossa NP, Strbian D, Tsivgoulis G, Turc G. European Stroke Organisation (ESO) guidelines on intravenous thrombolysis for acute ischaemic stroke.Eur Stroke J. 2021;6:I–LXII. doi: 10.1177/2396987321989865CrossrefGoogle Scholar
  • 11. Inzitari D, Giusti B, Nencini P, Gori AM, Nesi M, Palumbo V, Piccardi B, Armillis A, Pracucci G, Bono G, et al; MAGIC Study Group. MMP9 variation after thrombolysis is associated with hemorrhagic transformation of lesion and death.Stroke. 2013; 44:2901–2903. doi: 10.1161/STROKEAHA.113.002274LinkGoogle Scholar
  • 12. Wang X, Lee SR, Arai K, Tsuji K, Rebeck GW, Lo EH. Lipoprotein receptor-mediated induction of matrix metalloproteinase by tissue plasminogen activator.Nat Med. 2003; 9:1313–1317. doi: 10.1038/nm926CrossrefMedlineGoogle Scholar
  • 13. Benarroch EE. Tissue plasminogen activator: beyond thrombolysis.Neurology. 2007; 69:799–802. doi: 10.1212/01.wnl.0000269668.08747.78CrossrefMedlineGoogle Scholar
  • 14. Jickling GC, Liu D, Stamova B, Ander BP, Zhan X, Lu A, Sharp FR. Hemorrhagic transformation after ischemic stroke in animals and humans.J Cereb Blood Flow Metab. 2014; 34:185–199. doi: 10.1038/jcbfm.2013.203CrossrefMedlineGoogle Scholar
  • 15. Crucet M, Wust SJ, Spielmann P, Luscher TF, Wenger RH, Matter CM. Hypoxia enhances lipid uptake in macrophages: role of the scavenger receptors Lox1, SRA, and CD36.Atherosclerosis. 2013; 229:110–117. doi: 10.1016/j.atherosclerosis.2013.04.034CrossrefMedlineGoogle Scholar
  • 16. Hofmann A, Brunssen C, Wolk S, Reeps C, Morawietz H. Soluble LOX-1: a novel biomarker in patients with coronary artery disease, stroke, and acute aortic dissection?J Am Heart Assoc. 2020; 9:e013803. doi: 10.1161/JAHA.119.013803LinkGoogle Scholar
  • 17. Akhmedov A, Sawamura T, Chen CH, Kraler S, Vdovenko D, Lüscher TF. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1): a crucial driver of atherosclerotic cardiovascular disease.Eur Heart J. 2021; 42:1797–1807. doi: 10.1093/eurheartj/ehaa770CrossrefMedlineGoogle Scholar
  • 18. Sagar D, Gaddipati R, Ongstad EL, Bhagroo N, An LL, Wang J, Belkhodja M, Rahman S, Manna Z, Davis MA, et al. LOX-1: a potential driver of cardiovascular risk in SLE patients.PLoS One. 2020; 15:e0229184. doi: 10.1371/journal.pone.0229184CrossrefMedlineGoogle Scholar
  • 19. Grell AS, Frederiksen SD, Edvinsson L, Ansar S. Cerebrovascular gene expression in spontaneously hypertensive rats.PLoS One. 2017; 12:e0184233. doi: 10.1371/journal.pone.0184233CrossrefMedlineGoogle Scholar
  • 20. Grell AS, Mostajeran M, Frederiksen SD, Edvinsson L, Ansar S. Cerebrovascular gene expression in spontaneously hypertensive rats after transient middle cerebral artery occlusion.Neuroscience. 2017; 367:219–232. doi: 10.1016/j.neuroscience.2017.10.036CrossrefMedlineGoogle Scholar
  • 21. Rehnström M, Frederiksen SD, Ansar S, Edvinsson L. Transcriptome profiling revealed early vascular smooth muscle cell gene activation following focal ischemic stroke in female rats - comparisons with males.BMC Genomics. 2020; 21:883. doi: 10.1186/s12864-020-07295-2CrossrefMedlineGoogle Scholar
  • 22. Sawamura T, Kume N, Aoyama T, Moriwaki H, Hoshikawa H, Aiba Y, Tanaka T, Miwa S, Katsura Y, Kita T, et al. An endothelial receptor for oxidized low-density lipoprotein.Nature. 1997; 386:73–77. doi: 10.1038/386073a0CrossrefMedlineGoogle Scholar
  • 23. Li L, Renier G. The oral anti-diabetic agent, gliclazide, inhibits oxidized LDL-mediated LOX-1 expression, metalloproteinase-9 secretion and apoptosis in human aortic endothelial cells.Atherosclerosis. 2009; 204:40–46. doi: 10.1016/j.atherosclerosis.2008.08.008CrossrefMedlineGoogle Scholar
  • 24. Yoshimoto R, Fujita Y, Kakino A, Iwamoto S, Takaya T, Sawamura T. The discovery of LOX-1, its ligands and clinical significance.Cardiovasc Drugs Ther. 2011; 25:379–391. doi: 10.1007/s10557-011-6324-6CrossrefMedlineGoogle Scholar
  • 25. Tian K, Ogura S, Little PJ, Xu SW, Sawamura T. Targeting LOX-1 in atherosclerosis and vasculopathy: current knowledge and future perspectives.Ann N Y Acad Sci. 2019; 1443:34–53. doi: 10.1111/nyas.13984CrossrefMedlineGoogle Scholar
  • 26. Barreto J, Karathanasis SK, Remaley A, Sposito AC. Role of LOX-1 (Lectin-Like Oxidized Low-Density Lipoprotein Receptor 1) as a cardiovascular risk predictor: mechanistic insight and potential clinical use.Arterioscler Thromb Vasc Biol. 2021; 41:153–166. doi: 10.1161/ATVBAHA.120.315421LinkGoogle Scholar
  • 27. Morawietz H. LOX-1 and atherosclerosis: proof of concept in LOX-1-knockout mice.Circ Res. 2007; 100:1534–1536. doi: 10.1161/CIRCRESAHA.107.101105LinkGoogle Scholar
  • 28. Pirillo A, Norata GD, Catapano AL. LOX-1, OxLDL, and atherosclerosis.Mediators Inflamm. 2013; 2013:152786. doi: 10.1155/2013/152786CrossrefMedlineGoogle Scholar
  • 29. Vavere AL, Sinsakul M, Ongstad EL, Yang Y, Varma V, Jones C, Goodman J, Dubois VFS, Quartino AL, Karathanasis SK, et al. Lectin-like oxidized low-density lipoprotein receptor 1 inhibition in type 2 diabetes: phase 1 results.J Am Heart Assoc. 2023; 12:e027540. doi: 10.1161/JAHA.122.027540LinkGoogle Scholar
  • 30. Liang YQ, Kakino A, Matsuzaka Y, Mashimo T, Isono M, Akamatsu T, Shimizu H, Tajima M, Kaneko T, Li L, et al. LOX-1 (Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1) deletion has protective effects on stroke in the genetic background of stroke-prone spontaneously hypertensive rat.Stroke. 2020; 51:1835–1843. doi: 10.1161/STROKEAHA.120.029421LinkGoogle Scholar
  • 31. Markstad H, Edsfeldt A, Yao Mattison I, Bengtsson E, Singh P, Cavalera M, Asciutto G, Björkbacka H, Fredrikson GN, Dias N, et al. High levels of soluble lectinlike oxidized low-density lipoprotein receptor-1 are associated with carotid plaque inflammation and increased risk of ischemic stroke.J Am Heart Assoc. 2019; 8:e009874. doi: 10.1161/JAHA.118.009874LinkGoogle Scholar
  • 32. Lucero J, Suwannasual U, Herbert LM, McDonald JD, Lund AK. The role of the lectin-like oxLDL receptor (LOX-1) in traffic-generated air pollution exposure-mediated alteration of the brain microvasculature in Apolipoprotein (Apo) E knockout mice.Inhal Toxicol. 2017; 29:266–281. doi: 10.1080/08958378.2017.1357774CrossrefMedlineGoogle Scholar
  • 33. Greene C, Hanley N, Campbell M. Claudin-5: gatekeeper of neurological function.Fluids Barriers CNS. 2019; 16:3. doi: 10.1186/s12987-019-0123-zCrossrefMedlineGoogle Scholar
  • 34. Yuan S, Liu KJ, Qi Z. Occludin regulation of blood-brain barrier and potential therapeutic target in ischemic stroke.Brain Circ. 2020; 6:152–162. doi: 10.4103/bc.bc_29_20CrossrefMedlineGoogle Scholar
  • 35. Wang L, Chen Y, Feng D, Wang X. Serum ICAM-1 as a predictor of prognosis in patients with acute ischemic stroke.Biomed Res Int. 2021; 2021:5539304. doi: 10.1155/2021/5539304CrossrefMedlineGoogle Scholar
  • 36. Lindsberg PJ, Carpen O, Paetau A, Karjalainen-Lindsberg ML, Kaste M. Endothelial ICAM-1 expression associated with inflammatory cell response in human ischemic stroke.Circulation. 1996; 94:939–945. doi: 10.1161/01.cir.94.5.939LinkGoogle Scholar
  • 37. Lapchak PA, Chapman DF, Zivin JA. Metalloproteinase inhibition reduces thrombolytic (tissue plasminogen activator)-induced hemorrhage after thromboembolic stroke.Stroke. 2000; 31:3034–3040. doi: 10.1161/01.str.31.12.3034LinkGoogle Scholar
  • 38. Harada K, Suzuki Y, Yamakawa K, Kawakami J, Umemura K. Combination of reactive oxygen species and tissue-type plasminogen activator enhances the induction of gelatinase B in brain endothelial cells.Int J Neurosci. 2012; 122:53–59. doi: 10.3109/00207454.2011.623808CrossrefMedlineGoogle Scholar
  • 39. Sumii T, Lo EH. Involvement of matrix metalloproteinase in thrombolysis-associated hemorrhagic transformation after embolic focal ischemia in rats.Stroke. 2002; 33:831–836. doi: 10.1161/hs0302.104542LinkGoogle Scholar
  • 40. Arkelius K, Vivien D, Orset C, Ansar S. Validation of a stroke model in rat compatible with rt-PA-induced thrombolysis: new hope for successful translation to the clinic.Sci Rep. 2020; 10:12191. doi: 10.1038/s41598-020-69081-0CrossrefMedlineGoogle Scholar
  • 41. Lochhead JJ, Yang J, Ronaldson PT, Davis TP. Structure, function, and regulation of the blood-brain barrier tight junction in central nervous system disorders.Front Physiol. 2020; 11:914. doi: 10.3389/fphys.2020.00914CrossrefMedlineGoogle Scholar
  • 42. Miller DJ, Simpson JR, Silver B. Safety of thrombolysis in acute ischemic stroke: a review of complications, risk factors, and newer technologies.Neurohospitalist. 2011; 1:138–147. doi: 10.1177/1941875211408731CrossrefMedlineGoogle Scholar
  • 43. Sandercock P, Wardlaw JM, Lindley RI, Dennis M, Cohen G, Murray G, Innes K, Venables G, Czlonkowska A, Kobayashi A, et al; IST-3 collaborative group. The benefits and harms of intravenous thrombolysis with recombinant tissue plasminogen activator within 6 h of acute ischaemic stroke (the third international stroke trial [IST-3]): a randomised controlled trial.Lancet. 2012; 379:2352–2363. doi: 10.1016/S0140-6736(12)60768-5CrossrefMedlineGoogle Scholar
  • 44. Whiteley WN, Emberson J, Lees KR, Blackwell L, Albers G, Bluhmki E, Brott T, Cohen G, Davis S, Donnan G, et al; Stroke Thrombolysis Trialists’ Collaboration. Risk of intracerebral haemorrhage with alteplase after acute ischaemic stroke: a secondary analysis of an individual patient data meta-analysis.Lancet Neurol. 2016; 15:925–933. doi: 10.1016/S1474-4422(16)30076-XCrossrefMedlineGoogle Scholar
  • 45. Dong MX, Hu QC, Shen P, Pan JX, Wei YD, Liu YY, Ren YF, Liang ZH, Wang HY, Zhao LB, et al. Recombinant tissue plasminogen activator induces neurological side effects independent on thrombolysis in mechanical animal models of focal cerebral infarction: a systematic review and meta-analysis.PLoS One. 2016; 11:e0158848. doi: 10.1371/journal.pone.0158848CrossrefMedlineGoogle Scholar
  • 46. Wang X, Tsuji K, Lee SR, Ning M, Furie KL, Buchan AM, Lo EH. Mechanisms of hemorrhagic transformation after tissue plasminogen activator reperfusion therapy for ischemic stroke.Stroke. 2004; 35:2726–2730. doi: 10.1161/01.STR.0000143219.16695.afLinkGoogle Scholar
  • 47. Tsuji K, Aoki T, Tejima E, Arai K, Lee SR, Atochin DN, Huang PL, Wang X, Montaner J, Lo EH. Tissue plasminogen activator promotes matrix metalloproteinase-9 upregulation after focal cerebral ischemia.Stroke. 2005; 36:1954–1959. doi: 10.1161/01.STR.0000177517.01203.ebLinkGoogle Scholar
  • 48. Montaner J, Molina CA, Monasterio J, Abilleira S, Arenillas JF, Ribó M, Quintana M, Alvarez-Sabín J. Matrix metalloproteinase-9 pretreatment level predicts intracranial hemorrhagic complications after thrombolysis in human stroke.Circulation. 2003; 107:598–603. doi: 10.1161/01.cir.0000046451.38849.90LinkGoogle Scholar
  • 49. Wang W, Li M, Chen Q, Wang J. Hemorrhagic transformation after tissue plasminogen activator reperfusion therapy for ischemic stroke: mechanisms, models, and biomarkers.Mol Neurobiol. 2015; 52:1572–1579. doi: 10.1007/s12035-014-8952-xCrossrefMedlineGoogle Scholar
  • 50. Clark WM, Albers GW, Madden KP, Hamilton S. The rtPA (alteplase) 0- to 6-hour acute stroke trial, part A (A0276g): results of a double-blind, placebo-controlled, multicenter study. Thromblytic therapy in acute ischemic stroke study investigators.Stroke. 2000; 31:811–816. doi: 10.1161/01.str.31.4.811LinkGoogle Scholar
  • 51. Lin TC, Lee JD, Lin YH, Yuan RY, Weng HH, Huang YC, Lee M, Wu CY, Hsu HL, Hsu CY, et al. Timing of symptomatic infarct swelling following intravenous thrombolysis in acute middle cerebral artery infarction: a case-control study.Clin Appl Thromb Hemost. 2017; 23:814–820. doi: 10.1177/1076029616659693CrossrefMedlineGoogle Scholar
  • 52. Cuadrado E, Rosell A, Penalba A, Slevin M, Alvarez-Sabin J, Ortega-Aznar A, Montaner J. Vascular MMP-9/TIMP-2 and neuronal MMP-10 up-regulation in human brain after stroke: a combined laser microdissection and protein array study.J Proteome Res. 2009; 8:3191–3197. doi: 10.1021/pr801012xCrossrefMedlineGoogle Scholar
  • 53. Fugate JE, Rabinstein AA. Absolute and relative contraindications to IV rt-PA for acute ischemic stroke.Neurohospitalist. 2015; 5:110–121. doi: 10.1177/1941874415578532CrossrefMedlineGoogle Scholar
  • 54. Planas AM, Solé S, Justicia C. Expression and activation of matrix metalloproteinase-2 and -9 in rat brain after transient focal cerebral ischemia.Neurobiol Dis. 2001; 8:834–846. doi: 10.1006/nbdi.2001.0435CrossrefMedlineGoogle Scholar
  • 55. Ramos-Fernandez M, Bellolio MF, Stead LG. Matrix metalloproteinase-9 as a marker for acute ischemic stroke: a systematic review.J Stroke Cerebrovasc Dis. 2011; 20:47–54. doi: 10.1016/j.jstrokecerebrovasdis.2009.10.008CrossrefMedlineGoogle Scholar
  • 56. Lee CZ, Xue Z, Zhu Y, Yang GY, Young WL. Matrix metalloproteinase-9 inhibition attenuates vascular endothelial growth factor-induced intracerebral hemorrhage.Stroke. 2007; 38:2563–2568. doi: 10.1161/STROKEAHA.106.481515LinkGoogle Scholar
  • 57. Akhmedov A, Rozenberg I, Paneni F, Camici GG, Shi Y, Doerries C, Sledzinska A, Mocharla P, Breitenstein A, Lohmann C, et al. Endothelial overexpression of LOX-1 increases plaque formation and promotes atherosclerosis in vivo.Eur Heart J. 2014; 35:2839–2848. doi: 10.1093/eurheartj/eht532CrossrefMedlineGoogle Scholar
  • 58. Mehta JL, Sanada N, Hu CP, Chen J, Dandapat A, Sugawara F, Satoh H, Inoue K, Kawase Y, Jishage K, et al. Deletion of LOX-1 reduces atherogenesis in LDLR knockout mice fed high cholesterol diet.Circ Res. 2007; 100:1634–1642. doi: 10.1161/CIRCRESAHA.107.149724LinkGoogle Scholar
  • 59. Li D, Chen H, Romeo F, Sawamura T, Saldeen T, Mehta JL. Statins modulate oxidized low-density lipoprotein-mediated adhesion molecule expression in human coronary artery endothelial cells: role of LOX-1.J Pharmacol Exp Ther. 2002; 302:601–605. doi: 10.1124/jpet.102.034959CrossrefMedlineGoogle Scholar
  • 60. Li D, Mehta JL. Antisense to LOX-1 inhibits oxidized LDL-mediated upregulation of monocyte chemoattractant protein-1 and monocyte adhesion to human coronary artery endothelial cells.Circulation. 2000; 101:2889–2895. doi: 10.1161/01.cir.101.25.2889LinkGoogle Scholar
  • 61. Inoue K, Arai Y, Kurihara H, Kita T, Sawamura T. Overexpression of lectin-like oxidized low-density lipoprotein receptor-1 induces intramyocardial vasculopathy in apolipoprotein E-null mice.Circ Res. 2005; 97:176–184. doi: 10.1161/01.RES.0000174286.73200.d4LinkGoogle Scholar
  • 62. Chen J, Mehta JL, Haider N, Zhang X, Narula J, Li D. Role of caspases in Ox-LDL-induced apoptotic cascade in human coronary artery endothelial cells.Circ Res. 2004; 94:370–376. doi: 10.1161/01.RES.0000113782.07824.BELinkGoogle Scholar
  • 63. Ji KT, Qian L, Nan JL, Xue YJ, Zhang SQ, Wang GQ, Yin RP, Zhu YJ, Wang LP, Ma J, et al. Ox-LDL induces dysfunction of endothelial progenitor cells via activation of NF-κB.Biomed Res Int. 2015; 2015:175291. doi: 10.1155/2015/175291CrossrefMedlineGoogle Scholar
  • 64. Li D, Saldeen T, Romeo F, Mehta JL. Oxidized LDL upregulates angiotensin II type 1 receptor expression in cultured human coronary artery endothelial cells: the potential role of transcription factor NF-kappaB.Circulation. 2000; 102:1970–1976. doi: 10.1161/01.cir.102.16.1970LinkGoogle Scholar
  • 65. Bond M, Chase AJ, Baker AH, Newby AC. Inhibition of transcription factor NF-kappaB reduces matrix metalloproteinase-1, -3 and -9 production by vascular smooth muscle cells.Cardiovasc Res. 2001; 50:556–565. doi: 10.1016/s0008-6363(01)00220-6CrossrefMedlineGoogle Scholar
  • 66. Castier Y, Ramkhelawon B, Riou S, Tedgui A, Lehoux S. Role of NF-kappaB in flow-induced vascular remodeling.Antioxid Redox Signal. 2009; 11:1641–1649. doi: 10.1089/ars.2008.2393CrossrefMedlineGoogle Scholar
  • 67. Wang L, Deng L, Yuan R, Liu J, Li Y, Liu M. Association of matrix metalloproteinase 9 and cellular fibronectin and outcome in acute ischemic stroke: a systematic review and meta-analysis.Front Neurol. 2020; 11:523506. doi: 10.3389/fneur.2020.523506CrossrefMedlineGoogle Scholar
  • 68. Turner RJ, Sharp FR. Implications of MMP9 for blood brain barrier disruption and hemorrhagic transformation following ischemic stroke.Front Cell Neurosci. 2016; 10:56. doi: 10.3389/fncel.2016.00056CrossrefMedlineGoogle Scholar
  • 69. Barr TL, Latour LL, Lee KY, Schaewe TJ, Luby M, Chang GS, El-Zammar Z, Alam S, Hallenbeck JM, Kidwell CS, et al. Blood-brain barrier disruption in humans is independently associated with increased matrix metalloproteinase-9.Stroke. 2010; 41:e123–e128. doi: 10.1161/STROKEAHA.109.570515LinkGoogle Scholar
  • 70. Rosenberg GA, Yang Y. Vasogenic edema due to tight junction disruption by matrix metalloproteinases in cerebral ischemia.Neurosurg Focus. 2007; 22:E4. doi: 10.3171/foc.2007.22.5.5CrossrefMedlineGoogle Scholar
  • 71. Akhmedov A, Bonetti NR, Reiner MF, Spescha RD, Amstalden H, Merlini M, Gaul DS, Diaz-Cañestro C, Briand-Schumacher S, Spescha RS, et al. Deleterious role of endothelial lectin-like oxidized low-density lipoprotein receptor-1 in ischaemia/reperfusion cerebral injury.J Cereb Blood Flow Metab. 2019; 39:2233–2245. doi: 10.1177/0271678X18793266CrossrefMedlineGoogle Scholar
  • 72. Shen MY, Chen FY, Hsu JF, Fu RH, Chang CM, Chang CT, Liu CH, Wu JR, Lee AS, Chan HC, et al. Plasma L5 levels are elevated in ischemic stroke patients and enhance platelet aggregation.Blood. 2016; 127:1336–1345. doi: 10.1182/blood-2015-05-646117CrossrefMedlineGoogle Scholar
  • 73. Akamatsu T, Dai H, Mizuguchi M, Goto Y, Oka A, Itoh M. LOX-1 is a novel therapeutic target in neonatal hypoxic-ischemic encephalopathy.Am J Pathol. 2014; 184:1843–1852. doi: 10.1016/j.ajpath.2014.02.022CrossrefMedlineGoogle Scholar
  • 74. Fagan SC, Waller JL, Nichols FT, Edwards DJ, Pettigrew LC, Clark WM, Hall CE, Switzer JA, Ergul A, Hess DC. Minocycline to improve neurologic outcome in stroke (MINOS): a dose-finding study.Stroke. 2010; 41:2283–2287. doi: 10.1161/STROKEAHA.110.582601LinkGoogle Scholar
  • 75. Zhao JK, Guan FL, Duan SR, Zhao JW, Sun RH, Zhang LM, Wang DS. Effect of focal mild hypothermia on expression of MMP-9, TIMP-1, Tau-1 and β-APP in rats with cerebral ischaemia/reperfusion injury.Brain Inj. 2013; 27:1190–1198. doi: 10.3109/02699052.2013.804206CrossrefMedlineGoogle Scholar
  • 76. Xu S, Ogura S, Chen J, Little PJ, Moss J, Liu P. LOX-1 in atherosclerosis: biological functions and pharmacological modifiers.Cell Mol Life Sci. 2013; 70:2859–2872. doi: 10.1007/s00018-012-1194-zCrossrefMedlineGoogle Scholar
  • 77. Malhotra K, Chang JJ, Khunger A, Blacker D, Switzer JA, Goyal N, Hernandez AV, Pasupuleti V, Alexandrov AV, Tsivgoulis G. Minocycline for acute stroke treatment: a systematic review and meta-analysis of randomized clinical trials.J Neurol. 2018; 265:1871–1879. doi: 10.1007/s00415-018-8935-3CrossrefMedlineGoogle Scholar
  • 78. Pawletko K, Jędrzejowska-Szypułka H, Bogus K, Pascale A, Fahmideh F, Marchesi N, Grajoszek A, Gendosz de Carrillo D, Barski JJ. After ischemic stroke, minocycline promotes a protective response in neurons via the RNA-binding protein HuR, with a positive impact on motor performance.Int J Mol Sci. 2023; 24:9446. doi: 10.3390/ijms24119446CrossrefMedlineGoogle Scholar
  • 79. Hirschler L, Debacker CS, Voiron J, Köhler S, Warnking JM, Barbier EL. Interpulse phase corrections for unbalanced pseudo-continuous arterial spin labeling at high magnetic field.Magn Reson Med. 2018; 79:1314–1324. doi: 10.1002/mrm.26767CrossrefMedlineGoogle Scholar
  • 80. Choi-Lundberg DL, Lin Q, Schallert T, Crippens D, Davidson BL, Chang YN, Chiang YL, Qian J, Bardwaj L, Bohn MC. Behavioral and cellular protection of rat dopaminergic neurons by an adenoviral vector encoding glial cell line-derived neurotrophic factor.Exp Neurol. 1998; 154:261–275. doi: 10.1006/exnr.1998.6887CrossrefMedlineGoogle Scholar
  • 81. Encarnacion A, Horie N, Keren-Gill H, Bliss TM, Steinberg GK, Shamloo M. Long-term behavioral assessment of function in an experimental model for ischemic stroke.J Neurosci Methods. 2011; 196:247–257. doi: 10.1016/j.jneumeth.2011.01.010CrossrefMedlineGoogle Scholar
  • 82. Matthes F, Matuskova H, Arkelius K, Ansar S, Lundgaard I, Meissner A. An improved method for physical separation of cerebral vasculature and parenchyma enables detection of blood-brain-barrier dysfunction.NeuroSci. 2021; 2:59–74. doi: 10.3390/neurosci2010004CrossrefGoogle Scholar
  • 83. Wendt TS, Li YJ, Gonzales RJ. Ozanimod, an S1PR1 ligand, attenuates hypoxia plus glucose deprivation-induced autophagic flux and phenotypic switching in human brain VSM cells.Am J Physiol Cell Physiol. 2021; 320:C1055–C1073. doi: 10.1152/ajpcell.00044.2021CrossrefMedlineGoogle Scholar
  • 84. Wendt TS, Gonzales RJ. Ozanimod differentially preserves human cerebrovascular endothelial barrier proteins and attenuates MMP-9 activity following in vitro acute ischemic injury.Am J Physiol Cell Physiol. 2023; 325:C951–C971. doi: 10.1152/ajpcell.00342.2023CrossrefMedlineGoogle Scholar
  • 85. Orset C, Arkelius K, Anfray A, Warfvinge K, Vivien D, Ansar S. Combination treatment with U0126 and rt-PA prevents adverse effects of the delayed rt-PA treatment after acute ischemic stroke.Sci Rep. 2021; 11:11993. doi: 10.1038/s41598-021-91469-9CrossrefMedlineGoogle Scholar

eLetters(0)

eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. Authors of the article cited in the comment will be invited to reply, as appropriate.

Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page.