Elena Meseguer
email: elena.meseguer@aphp.fr

Devy Diallo
email: devy.diallo@gmail.com

Julien Labreuche
email: julien.labreuche.chru@gmail.com

Hugo Charles

Sandrine Delbosc
email: sandrine.delbosc@inserm.fr

Gabrielle Mangin
email: gabrielle.mangin@inserm.fr

Linsay Monteiro Tavares

Giuseppina Caligiuri
email: giuseppina.caligiuri@inserm.fr

Antonino Nicoletti
email: antonino.nicoletti@inserm.fr

Pierre Amarenco
email: pierre.amarenco@aphp.fr

Osteopontin Predicts Three-Month Outcome in Stroke Patients Treated by Reperfusion Therapies

Keywords: acute stroke, biomarkers, assessment, outcomes, thrombolytic therapy, thrombectomy

Establishing a prognosis at hospital admission after stroke is a major challenge. Inflammatory processes, hemostasis, vascular injury, and tissue remodeling are all involved in the early response to stroke. This study analyzes whether 22 selected biomarkers, sampled at admission, predict clinical outcomes in 153 stroke patients treated by thrombolysis and mechanical endovascular treatment (MET). Biomarkers were related to hemostasis (u-plasminogen activator/urokinase (uPA/urokinase), serpin E1/PAI-1, serpin C1/antithrombin-III, kallikrein 6/neurosin, alpha 2-macroglobulin), inflammation[myloperoxidase (MPO), chemokine ligand 2/monocyte chemoattractant protein-1 chemokine (CCL2/MCP-1), adiponectin, resistin, cell-free DNA (cDNA), CD40 Ligand (CD40L)], endothelium activation (Vascular cell adhesion protein 1 (VCAM-1) intercellular adhesion molecule 1 (ICAM-1), platelet endothelial cell adhesion molecule 1 (CD31/PECAM-1)], and tissue remodeling (total cathepsin S, osteopontin, cystatin C, neuropilin-1, matrix metallopeptidase 2 (MMP-2), matrix metallopeptidase 3 (MMP-3), matrix metallopeptidase 9 (MMP-9), matrix metallopeptidase 13 (MMP-13)]. Correlations between their levels and excellent neurological improvement (ENI) at 24 h and good outcomes (mRS 0-2) at three months were tested. Osteopontin and favorable outcomes reached the significance level (p = 0.008); the adjusted OR per SD increase in log-transformed osteopontin was 0.34 (95% CI, 0.18-0.62). The relationship between total cathepsin S and MPO with ENI, was borderline of significance (p = 0.064); the adjusted OR per SD increase in log-transformed of total cathepsin S and MPO was 0.54 (95% CI, 0.35-0.81) and 0.51 (95% CI, 0.32-0.80), respectively. In conclusion, osteopontin levels predicted three-month favorable outcomes, supporting the use of this biomarker as a complement of clinical and radiological parameters for predicting stroke prognosis.

Introduction

The Global Burden of Disease estimated that one person in four aged 25 years will have a stroke in the rest of her/his life [START_REF] Gbd ; Feigin | Regional, and Country-Specific Lifetime Risks of Stroke, 1990 and 2016[END_REF]. Among them, ischemic stroke represents 80%. Stroke accounts for almost 5% of all disability-adjusted life-years and 10% of all deaths worldwide [START_REF] Gbd ; Feigin | Regional, and Country-Specific Lifetime Risks of Stroke, 1990 and 2016[END_REF]. These numbers make stroke a major healthcare problem all over the world.

Experimental Section

Patients

We conducted a prospective study with patients admitted for ischemic stroke who received thrombolysis and/or MET within 6 h of symptom onset at Bichat Hospital, Paris, France. Only patients arriving within working hours of the research nurse were included in the study. Plasma samples were taken from all patients at arrival, before any treatment was given. We collected data from each patient on demographics, risk factors, previous medications for stroke, baseline National Institutes of Health Stroke Scale (NIHSS) [START_REF] Brott | Measurements of acute cerebral infarction: A clinical examination scale[END_REF], and blood pressure at admission. All patients had brain imaging to support the diagnosis. Patients with ischemic stroke were treated with iv recombinant tissue plasminogen activator (rtPA), and in the case of documented arterial occlusion EVT, including intra-arterial (IA) thrombolysis +/-MET was performed.

Data Collection and Definitions

Data were collected using a structured questionnaire. Smoking was classified as current (any smoking within the past six months), former (more than six months since cessation), or never smokers. Hypertension and hypercholesterolemia were defined by treatment history. Subjects were classified as diabetic when treated for type 1 or type 2 diabetes. The stroke severity was assessed using the NIHSS score at admission. The arterial status of the occluded artery was monitored with conventional angiography during the endovascular procedure and measured using the Thrombolysis in Cerebral Infarction (TICI) score [START_REF] Zaidat | Recommendations on Angiographic Revascularization Grading Standards for Acute Ischemic Stroke[END_REF]. All patients underwent a computed tomography (CT) or magnetic resonance imaging (MRI) scan 24 h after treatment onset, to assess hemorrhagic complications. Intracranial hemorrhage was defined and classified according to the European Co-operative Acute Stroke Study III (ECASS-III) trial [START_REF] Hacke | Thrombolysis with Alteplase 3 to 4.5 Hours after Acute Ischemic Stroke[END_REF]. Functional outcomes were assessed at three months using the modified Rankin Scale (mRS) [START_REF] Van Swieten | Interobserver agreement for the assessment of handicap in stroke patients[END_REF] during face-to-face interviews or via telephone calls by a senior vascular neurologist (E.M) certified for mRS evaluation.

All participants provided informed consent. This study was performed according to the principles of the Declaration of Helsinki (1964). Informed consent was obtained from the patient or surrogate, and the research protocol was approved by the Ethics Committee from the Ambroise Pare Hospital, on 23 September 2008, code ID RCB:2008-A00966-49 and 5 January 2010, code ID RCB: 2009-A01165-52, Paris, France.

Outcome Measurements

A favorable outcome was defined as an mRS score of ≤2 at 90 days. An excellent favorable clinical outcome was defined as an mRS score of ≤1 at 90 days. Early neurological improvement (ENI) was defined as an improvement of 4 or more points on this scale (compared with baseline) 24 h after thrombolytic/EVT treatment or NIHSS of 0 24 h after thrombolytic/EVT treatment.

In the case of EVT, the recanalization rate was measured using the TICI at the end of the endovascular procedure. Successful recanalization was defined as TICI 2B or 3 [START_REF] Zaidat | Recommendations on Angiographic Revascularization Grading Standards for Acute Ischemic Stroke[END_REF]. Hemorrhagic transformation was defined as the appearance of hemorrhagic transformation at 24 h on CT or MRI. Symptomatic hemorrhagic transformation was defined according to the ECASS-III definition as the presence of any hemorrhage on the follow-up CT/MRI imaging scan associated with an increase of 4 points in NIHSS score or death within 7 days [START_REF] Hacke | Thrombolysis with Alteplase 3 to 4.5 Hours after Acute Ischemic Stroke[END_REF].

Blood Biomarkers

Patients were sampled within 6 h after stroke onset and before any reperfusion treatment was given. We selected 22 biomarkers according to their main functions in ischemic stroke:

Hemostasis: u-plasminogen activator/urokinase (uPA)/urokinase, serpin E1/PAI-1, serpin C1/antithrombin-III, kallikrein 6/neurosin, alpha 2-macroglobulin. Inflammation: Myloperoxidase (MPO), chemokine ligand 2/monocyte chemoattractant protein-1 chemokine (CCL2/MCP-1), adiponectin, resistin, cell-free DNA (c-DNA), CD40 Ligand (CD40L). Endothelium activation: Vascular cell adhesion protein 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), platelet endothelial cell adhesion molecule 1 (PECAM-1 or CD31). Tissue remodeling: Total cathepsin S, osteopontin, cystatin C, neuropilin-1, MMP-2, matrix metallopeptidase 2 (MMP-2), matrix metallopeptidase 3 (MMP-3), matrix metallopeptidase 9 (MMP-9), matrix metallopeptidase 13 (MMP-13).

The concentration of biomarkers, except c-DNA, was measured in plasma, collected on EDTA tubes, by using commercials human Magnetic Luminex assay kits (reference LXSAHM, R&D system Bio-Techne) according to the manufacturer's instruction. Briefly, 50 µL of diluted plasma or standard was incubated with magnetic beads (pre-coated with analyte-specific antibodies). After different steps of washing, samples or standards were incubated with biotinylated antibodies specific to the analytes of interest. Following a wash to remove any unbound biotinylated antibody, streptavidin-phycoerythrin conjugate (Streptavidin-PE), which binds to the biotinylated antibody, was added to samples or standards, and after final washes, the fluorescence intensity of the magnetic beads was detected on a Bio-Plex 200 system (Bio-rad). The minimum detection limit in ng/mL is for uPA/urokinase 0.12, serpin E1/PAI-1 0.02, serpin C1/antithrombin-III 2.84, total cathepsin S 0.09, CCL2/MCP-1 0.03, CD31/PECAM-1 0.68, CD40L 0.21, ICAM-1 6.89, kallikrein 6/neurosin 0.28, MMP-3 0.08, MMP-13 0.12, osteopontin 1.8, resistin 0.04, VCAM-1 7.80, alpha 2-macroglobulin 1.66, adiponectin 0.95, cystatin C 0.66, MMP-9 0.13, neuropilin-1 0.08, MMP-2 0.32, MPO 0.13.

The c-DNA concentration was determined in plasma EDTA, using QuantitTM PicogreenH dsDNA Reagent (Invitrogen). Briefly, 10 µL of samples and Lambda DNA standard (1 ng/mL -1 mg/mL) were diluted in TE buffer (200 mM Tris-HCl, 20 mM EDTA, pH 7.5, 100 mL final) before addition of 100 µL PicogreenH dsDNA reagent. After mixing, and incubation for 5 min at room temperature in the dark, the fluorescence was measured using a microplate reader (excitation 480 nm, emission 520 nm).

Statistics

Quantitative variables are expressed as mean (standard deviation) in the case of normal distribution or median (interquartile range) otherwise. Categorical variables are expressed as numbers (percentages). The normality of distributions was assessed using histograms and the Shapiro-Wilk test. To assess the selection bias, baseline characteristics were described according to the included and non-included patients, and the magnitude of the between-group differences was assessed by calculating the absolute standardized differences; an absolute standardized difference <0.2 was interpreted as a small difference [START_REF] Cohen | Statistical Power Analysis for the Behavioral Sciences[END_REF]. Baseline characteristics were further described and compared according to the pre-specified primary outcome (favorable outcome) using the Chi-square test or Fisher's exact test for categorical variables, and Student's t-test or Mann-Whitney U-test for quantitative variables according to the normality of distributions. We firstly assessed the associations of blood biomarkers with favorable, excellent, and ENI outcomes in bivariate analyses by using Student's t-test on log-transformed (to reduce the skewness of distribution) for all blood biomarkers (except for Alpha2 macroglobulin). Associations of blood biomarkers with each outcome were further investigated in multivariate logistic regression analysis adjusted for the following pre-specified confounders: age, admission NIHSS, documented occlusion (none vs. Internal Carotid Artery (ICA) occlusion vs. others arterial occlusion), and AIS treatments. Strength of associations was assessed by calculating the adjusted odds ratios (ORs) and theirs 95% confidence intervals (CIs) per one SD increase in blood biomarkers. Log-linearity assumptions were checked using restricted cubic spline functions [START_REF] Harrell | Multivariable prognostic models: Issues in developing models, evaluating assumptions and adequacy, and measuring and reducing errors[END_REF]. p-values for bivariate and multivariate analyses were corrected for multiple comparisons (22 biomarkers tested) by using the Holm-Bonferroni method. Statistical testing was done at the two-tailed α level of 0.05. Data were analyzed using the SAS software package, release 9.4 (SAS Institute, 100 SAS Campus Drive Cary, NC 27513-2414, Cary, NC, USA).

Results

Study Sample

From January 2009 to June 2013, 618 patients with AIS within 6 h of symptom onset were consecutively treated by AIS treatment (thrombolysis and/or EVT) at our center. Of these patients, 151 patients had a blood sample taken just prior to acute treatment with 90-day mRs evaluation and were included in the present study (Figure 1). compared according to the pre-specified primary outcome (favorable outcome) using the Chi-square test or Fisher's exact test for categorical variables, and Student's t-test or Mann-Whitney U-test for quantitative variables according to the normality of distributions. We firstly assessed the associations of blood biomarkers with favorable, excellent, and ENI outcomes in bivariate analyses by using Student's t-test on log-transformed (to reduce the skewness of distribution) for all blood biomarkers (except for Alpha2 macroglobulin). Associations of blood biomarkers with each outcome were further investigated in multivariate logistic regression analysis adjusted for the following pre-specified confounders: age, admission NIHSS, documented occlusion (none vs. Internal Carotid Artery (ICA) occlusion vs. others arterial occlusion), and AIS treatments. Strength of associations was assessed by calculating the adjusted odds ratios (ORs) and theirs 95% confidence intervals (CIs) per one SD increase in blood biomarkers. Log-linearity assumptions were checked using restricted cubic spline functions [START_REF] Harrell | Multivariable prognostic models: Issues in developing models, evaluating assumptions and adequacy, and measuring and reducing errors[END_REF]. P-values for bivariate and multivariate analyses were corrected for multiple comparisons (22 biomarkers tested) by using the Holm-Bonferroni method. Statistical testing was done at the two-tailed α level of 0.05. Data were analyzed using the SAS software package, release 9.4 (SAS Institute, 100 SAS Campus Drive Cary, NC 27513-2414, Cary, NC, USA).

Results

Study Sample

From January 2009 to June 2013, 618 patients with AIS within 6 h of symptom onset were consecutively treated by AIS treatment (thrombolysis and/or EVT) at our center. Of these patients, 151 patients had a blood sample taken just prior to acute treatment with 90-day mRs evaluation and were included in the present study (Figure 1). The median age of the study sample was 73 years (IQR, 58 to 83), and 51% of them were men. The median NIHSS was 13 (IQR, 5 to 18), and 31% of patients had no documented occlusion. 68 patients were treated by IV thrombolysis, 55 by an IV + EVT approach (ia thrombolysis n = 21 and ia thrombolysis + adjunctive MET, n = 34), and 28 by an exclusively EVT (ia thrombolysis, n = 8; MET, n = 15; ia thrombolysis + adjunctive MET, n = 5). Baseline characteristics for included and excluded patients are available in Table S1; except for diabetes (less frequent in included patients), admission NIHSS (higher in included patients), documented occlusion (more frequent in included patients), and AIS treatment (more frequent use of an endovascular approach in included patients, especially The median age of the study sample was 73 years (IQR, 58 to 83), and 51% of them were men. The median NIHSS was 13 (IQR, 5 to 18), and 31% of patients had no documented occlusion. 68 patients were treated by IV thrombolysis, 55 by an IV + EVT approach (ia thrombolysis n = 21 and ia thrombolysis + adjunctive MET, n = 34), and 28 by an exclusively EVT (ia thrombolysis, n = 8; MET, n = 15; ia thrombolysis + adjunctive MET, n = 5). Baseline characteristics for included and excluded patients are available in Table S1; except for diabetes (less frequent in included patients), admission NIHSS (higher in included patients), documented occlusion (more frequent in included patients), and AIS treatment (more frequent use of an endovascular approach in included patients, especially MET), baseline characteristics were well balanced (standardized difference <20%).

In study sample, 72 patients (47.7%; 95% CI, 39.7-55.7) had an ENI, 64 (42.4%; 95% CI, 34.5-50.3) had an excellent functional outcome and 80 (53.0%; 95% CI, 45.0-60.9) had a favorable outcome. Baseline characteristics of patients with and without favorable outcomes (pre-specified as primary outcomes) are reported in Table 1. Patients with favorable outcomes were significantly younger, had less often pre-stroke mRS >1, a lower glucose level at admission, a lower NIHSS score at admission, less often documented occlusion, and were consequently more often treated by IV fibrinolysis alone.

Blood Biomarkers

Distribution of the 22 biomarkers tested (five for hemostasis, six for inflammation, three for endothelial activation, and eight for tissue remodeling) are reported in Table S2.

Hemostasis Biomarkers and Outcomes

As shown in Tables 234, in bivariable analysis corrected for multiple comparisons, none of the hemostasis biomarkers were associated with any outcomes. Similar results were found in a multivariable analysis adjusted for pre-specified confounders (age, admission NIHSS, documented occlusion, and AIS treatment). Values are median (IQR) except for Alpha2 macroglobulin, where mean (SD) is reported. For all biological markers (except Alpha2 macroglobulin), p-values and ORs were calculated on log-transformed values. ORs were calculated per one SD increase in biological markers. An excellent outcome is defined as 90-day mRS ≤ 1 or equal to pre-stroke mRS. 1 corrected for multiple comparisons using Holm Bonferroni method. 2 adjusted for age, admission NIHSS, documented occlusion (none vs. ICA occlusion vs. others arterial occlusion), and AIS treatments (including IV +-EVT). Abbreviations: AIS = acute ischemic stroke; CI = confidence interval; IV Intra-venous, EVT Endovascular treatment; ICA = internal carotid artery; IQR = interquartile range; IV = intravenous; MET = mechanical endovascular treatment; mRS = modified Rankin Scale; OR = odds ratio; SD = standard deviation.

Inflammation Biomarkers and Outcomes

Of the six inflammation biomarkers, only MPO was associated with each study outcome in the bivariable analysis (p corrected for multiple comparisons <0.01). MPO was lower in patients with favorable outcomes than those without favorable outcomes (median (IQR), 38 vs. 51 ). Similarly, an increased MPO level was associated with a reduced likelihood of excellent (Table 3) and ENI outcomes (Table 4). In a multivariable analysis adjusted for pre-specified confounders, MPO remained associated with reduced likelihood of each outcome before, but not after correction for multiple comparisons; the adjusted OR per one SD increase in log-transformed MPO was 0.51 (95% CI, 0.32 to 0.89) for a favorable outcome, 0.60 (95% CI, 0.37 to 0.97) for an excellent outcome, and 0.51 (95% CI, 0.32 to 0.80) for ENI. Values are median (IQR) except for Alpha2 macroglobulin, where mean (SD) is reported. For all biological markers (except Alpha2 macroglobulin), p-values and ORs were calculated on log-transformed values. ORs were calculated per one SD increase in biological markers. An early neurological outcome is defined as 90-day mRS ≤ 1 or equal to pre-stroke mRS. 1 corrected for multiple comparisons using Holm Bonferroni method. 2 adjusted for age, admission NIHSS, documented occlusion (none vs. ICA occlusion vs. others arterial occlusion), and AIS treatments (including IV+-EVT). Abbreviations: AIS = acute ischemic stroke; CI = confidence interval; IV Intra-venous, EVT Endovascular thrombectomy; ICA = internal carotid artery; IQR = interquartile range; IV = intravenous; MET = mechanical endovascular treatment; mRS = modified Rankin Scale; OR = odds ratio; SD = standard deviation.

Endothelial Activation Biomarkers and Outcomes

In bivariable analysis corrected for multiple comparisons, only VCAM-1 was significantly associated with a favorable and excellent outcomes, but not with ENI. Similar to MPO, increased VCAM-1 level was associated with a reduced likelihood of favorable and excellent in multivariate analysis before, but not after correction for multiple comparisons (Tables 2 and3); the adjusted OR per one SD increase in log-transformed VCAM-1 was 0.53 (95% CI, 0.31 to 0.90) for a favorable outcome and 0.54 (95% CI, 0.33 to 0.89) for an excellent outcome.

Tissue Remodeling Biomarkers and Outcomes

Of the eight tissue remodeling biomarkers, a favorable outcome was significantly associated with lower osteopontin, and neuropilin-1 levels in bivariable analysis corrected for multiple comparisons (Table 2); a similar association (but not significant) was observed for total cathepsin S and cystatin C. Regarding other outcomes, total cathepsin S, osteopontin and neuropilin-1 were associated with reduced likelihood of excellent outcomes (Table 3), and only total cathepsin S was associated with reduced likelihood of ENI (Table 4). In a multivariable analysis, total cathepsin S and osteopontin were associated with reduced likelihood of functional outcomes, but only the association between osteopontin and the favorable outcome reached the significance level after multiple comparison correction (p = 0.008); the adjusted OR per SD increase in log-transformed osteopontin was 0.34 (95% CI, 0.18 to 0.62) for a favorable outcome and 0.49 (95% CI, 0.29 to 0.81) for an excellent outcome. The association of total cathepsin S with ENI was of borderline significance in a multivariable analysis corrected for multiple comparisons with an OR per SD increase in log-transformed total cathepsin S of 0.54 (95% CI, 0.35 to 0.81).

Osteopontin and Favorable Outcomes According to AIS Treatment

As shown in Figure 2, there is no evidence of heterogeneity in association with osteopontin levels and a favorable outcome, according to AIS treatment. In patients under exclusively IV thrombolysis and those treated by a combination of IV thrombolysis and EVT; osteopontin was related to poor outcomes (p = 0.014 and p = 0.007, respectively). In those under EVT (including IA thrombolysis and/or MET), there was no difference (p = 0.13), but only six patients with favorable outcomes were included in the analyses (Figure 2A). Osteopontin levels were also related to favorable outcomes in patients who received MET (p = 0.026) and those who not (p = 0.001) (Figure 2B).

Discussion

Previous studies in ischemic stroke have correlated blood biomarkers to clinical presentation [START_REF] Montaner | A panel of biomarkers including caspase-3 and D-dimer may differentiate acute stroke from stroke-mimicking conditions in the emergency department[END_REF], outcomes [START_REF] Zhong | Multiple biomarkers covering distinct pathways for predicting outcomes after ischemic stroke[END_REF], and hemorrhagic transformation [START_REF] Lu | Potential biomarkers for predicting hemorrhagic transformation of ischemic stroke[END_REF]. In our study, low levels in plasma EDTA of circulating osteopontin, a tissue remodeling biomarker, predicted favorable outcomes at three months in adjusted and unadjusted analyses. Moreover, cathepsin S and MPO levels in plasma EDTA were predictors in non-adjusted analyses of ENI.

Discussion

Previous studies in ischemic stroke have correlated blood biomarkers to clinical presentation [START_REF] Montaner | A panel of biomarkers including caspase-3 and D-dimer may differentiate acute stroke from stroke-mimicking conditions in the emergency department[END_REF], outcomes [START_REF] Zhong | Multiple biomarkers covering distinct pathways for predicting outcomes after ischemic stroke[END_REF], and hemorrhagic transformation [START_REF] Lu | Potential biomarkers for predicting hemorrhagic transformation of ischemic stroke[END_REF]. In our study, low levels in plasma EDTA of circulating osteopontin, a tissue remodeling biomarker, predicted favorable outcomes at three months in adjusted and unadjusted analyses. Moreover, cathepsin S and MPO levels in plasma EDTA were predictors in non-adjusted analyses of ENI.

Patients with lower levels of osteopontin did better at three months than those with higher levels. It was the only biomarker that predicted favorable outcomes at three months in adjusted and unadjusted analyses, and it also predicted excellent outcomes in unadjusted analyses. These results were independent of the revascularization treatment. Osteopontin is an acidic phosphoglycoprotein that was first described as being secreted by transformed cells isolated from bone [START_REF] Butler | The Nature and Significance of Osteopontin[END_REF][START_REF] Butler | Structural and Functional Domains of Osteopontin[END_REF]. It is upregulated in response to injury, stress, and inflammation in diverse cells [START_REF] Lok | Osteopontin in Vascular Disease[END_REF]. This protein is a potent mediator in the development and atherosclerosis progression [START_REF] Isoda | Osteopontin transgenic mice fed a high-cholesterol diet develop early fatty-streak lesions[END_REF], and is involved in hemostasis, angiogenesis, wound healing, and immune responses [START_REF] Myles | Thrombin Hydrolysis of Human Osteopontin Is Dependent on Thrombin Anion-binding Exosites[END_REF][START_REF] Kumar | Functional Characterization of Stromal Osteopontin in Melanoma Progression and Metastasis[END_REF][START_REF] Mckee | Secretion of osteopontin by macrophages and its accumulation at tissue surfaces during wound healing in mineralized tissues: A potential requirement for macrophage adhesion and phagocytosis[END_REF][START_REF] Wang | Osteopontin: Role in immune regulation and stress responses[END_REF]. In the brain, OPN is expressed constitutively and is upregulated upon experimental cerebral ischemia [START_REF] Ellison | Osteopontin and its Integrin Receptor αvβ3Are Upregulated During Formation of the Glial Scar After Focal Stroke[END_REF][START_REF] Lee | Transient upregulation of osteopontin mRNA in hippocampus and striatum following global forebrain ischemia in rats[END_REF], where it has been shown to play a role in remodeling processes by modulating macrophage recruitment, matrix repair, astroglial cell migration, and gliosis. In accordance with our results, a previous study in patients treated by IV thrombolysis has found that high initial levels of osteopontin were related to bad outcomes at three months [START_REF] Mendioroz | Osteopontin predicts long-term functional outcome among ischemic stroke patients[END_REF]. Moreover, another study has shown that high serum levels of osteopontin seven days after ischemic stroke, were positively correlated with cerebral infarction volume and bad neurological status at 3-month [START_REF] Carbone | Serum osteopontin levels are upregulated and predict disability after an ischaemic stroke[END_REF]. High osteopontin levels were also related to stroke recurrence in the SPARCL Trial [START_REF] Ganz | Association of Osteopontin, Neopterin, and Myeloperoxidase with Stroke Risk in Patients with Prior Stroke or Transient Ischemic Attacks[END_REF] and bad outcomes, in the case of stroke recurrence in this trial (unpublished data). All these data points at osteopontin as a key informative biomarker in the case of ischemic stroke.

We also found that circulating levels of cathepsin S were associated with reduced likelihood of excellent outcomes and of ENI, but it must be noted that statistical significance was lost after multiple comparison correction. Cathepsins are proteases that promote the degradation of damaged proteins in the lysosome, and they are also secreted extracellularly in a variety of pathological processes, including arthritis, cancer, and cardiovascular disease [START_REF] Wilkinson | Therapeutic, diagnostic, and prognostic potential[END_REF]. Specifically, cathepsin S has been implicated in elastin degradation in cardiovascular disease, and high levels of cathepsin S have been described in the atheroma plaque [START_REF] Sukhova | Expression of the elastolytic cathepsins S and K in human atheroma and regulation of their production in smooth muscle cells[END_REF]. Experimental animal studies have shown that cathepsin S promotes endothelial cell invasion and neovessel growth [START_REF] Shi | Deficiency of the Cysteine Protease Cathepsin S Impairs Microvessel Growth[END_REF]. To the best of our knowledge, the role of cathepsin S in ischemic stroke has not yet been studied. Our study suggests that this biomarker could be informative for the clinical evolution at 24 h, but more studies are needed to confirm this hypothesis.

MPO levels were also borderline as a predictor of a reduced likelihood of ENI. MPO is produced mostly by polymorphonuclear neutrophils [START_REF] Lau | Myeloperoxidase and its contributory role in inflammatory vascular disease[END_REF]. MPO is stored in cytoplasmic membrane-bound azurophilic granules, which can be secreted out to the extracellular space by degranulation or exocytosis upon oxidative stress and different inflammatory responses. Myeloperoxidase is the only type of peroxidase that uses H2O2 to form different hypohalous acids [START_REF] Davies | Myeloperoxidase-derived oxidation: Mechanisms of biological damage and its prevention[END_REF]. Through its activities, MPO increases tissue injury [START_REF] Nussbaum | Myeloperoxidase: A Leukocyte-Derived Protagonist of Inflammation and Cardiovascular Disease[END_REF]. In an experimental model of cerebral ischemia-reperfusion in rats, MPO levels were found to be significantly increased in the cerebral cortex 24 h after reperfusion, and brain MPO activity correlated with the appearance of neutrophils. Neutrophil depletion prevented increased MPO activity and reduced infarct size [START_REF] Matsuo | Correlation between myeloperoxidase-quantified neutrophil accumulation and ischemic brain injury in the rat. Effects of neutrophil depletion[END_REF]. MPO knockout mice subjected to the same stroke model also showed a similar reduction in the final lesion volume [START_REF] Forghani | Myeloperoxidase Propagates Damage and is a Potential Therapeutic Target for Subacute Stroke[END_REF]. Human studies showed that patients with ischemic stroke display higher serum levels of MPO compared to healthy subjects [START_REF] Palm | Serum matrix metalloproteinase-8, tissue inhibitor of metalloproteinase and myeloperoxidase in ischemic stroke[END_REF], and higher levels of MPO are related to higher NIHSS [START_REF] Palm | Serum matrix metalloproteinase-8, tissue inhibitor of metalloproteinase and myeloperoxidase in ischemic stroke[END_REF] and mortality [START_REF] Tay | Serum myeloperoxidase levels in predicting the severity of stroke and mortality in acute ischemic stroke patients[END_REF]. These data might indicate an early deleterious involvement of neutrophils, since high levels of circulating MPO level is a marker of increased neutrophil activity. In line with this hypothesis, higher neutrophil counts before thrombolysis for cerebral ischemia predicted worse outcomes [START_REF] Maestrini | Higher neutrophil counts before thrombolysis for cerebral ischemia predict worse outcomes[END_REF]. Altogether, these reports and our study indicate that circulating MPO level taken as a surrogate marker of neutrophil activity is a useful biomarker for stroke prognosis.

Few limitations derive from the design of the present study. Patients were treated by IA thrombolysis and first-generation thrombectomy devices. Nowadays, devices display better performance, and higher levels of recanalization are achieved. We did not include patients treated by antithrombotic treatment; the interest of these biomarkers in this population is unknown. Samplings were from a single-center, and they were collected in a limited number of patients. These results should be taken as preliminary and exploratory, and need to be reproduced in larger, multicenter cohorts to be generalizable and to ultimately develop a point of care device. Yet, the objective of this study was to analyze the predictive value of biomarkers that had been only scarcely studied in the setting of patients treated with thrombolysis.

Conclusions

In conclusion, osteopontin predicted favorable outcomes at three-month, while cathepsin S and MPO were related to ENI in unadjusted analysis. No biomarker related to hemostasis or endothelial activation biomarker predicted ENI or three-month outcome. We did not find any biomarker related to excellent outcomes. Larger studies are warranted to confirm the role of these biomarkers as predictors of stroke outcome.

Figure 1 .

 1 Figure 1. Study flow-chart. AIS = Acute ischemic stroke.

Figure 1 .

 1 Figure 1. Study flow-chart. AIS = Acute ischemic stroke.

  ≤ 2 or equal to pre-stroke mRs. Arterial occlusion documented before AIS treatment by transcranial Doppler, MRA or CT angiography. Abbreviations: AIS = acute ischemic stroke; BP = blood pressure; CT = computed tomography; EVT = endovascular treatment; IA = intra-arterial; ICA = internal carotid artery; IQR = interquartile range; IV = intravenous; MCA = middle cerebral artery; MET = mechanical endovascular treatment; MRA = magnetic resonance angiography; mRS = modified Rankin Scale; NIHSS = National Institutes of Health Stroke Scale; SD = standard deviation.

Figure 2 .

 2 Figure 2. (A) According to treatment strategy. (B) According to mechanical endovascular treatment (MET). Distribution of osteopontin according to a favorable outcome and AIS treatment. Boxes show the 25th, 50th, and 75th percentiles, and unadjusted p-values (Student's t-test on log-transformed values) are reported. Abbreviations: EVT = endovascular treatment; IV = intravenous thrombolysis; MET = mechanical endovascular treatment.

Figure 2 .

 2 Figure 2. (A) According to treatment strategy. (B) According to mechanical endovascular treatment (MET). Distribution of osteopontin according to a favorable outcome and AIS treatment. Boxes show the 25th, 50th, and 75th percentiles, and unadjusted p-values (Student's t-test on log-transformed values) are reported. Abbreviations: EVT = endovascular treatment; IV = intravenous thrombolysis; MET = mechanical endovascular treatment.

Table 1 .

 1 Patients' and treatment characteristics according to a favorable outcome.

		Favorable Outcome
	Characteristics	No (n = 71)	Yes (n = 80)	p-Value
	Age, median (IQR)	78 (66 to 85)	64 (53 to 80)	<0.001
	Men	35/71 (49.3)	43/80 (53.8)	0.58
	Medical history
	Hypertension	46/71 (64.8)	45/78 (57.7)	0.37
	Hypercholesterolemia	27/70 (38.6)	30/77 (39.0)	0.96
	Diabetes	7/70 (10.0)	4/77 (5.2)	0.27
	Current smoking	11/67 (16.4)	17/77 (22.1)	0.39
	Antithrombotic medications	28/70 (40.0)	23/78 (29.5)	0.18
	Antiplatelet	21/70 (30.0)	18/78 (23.1)	0.34
	Anticoagulant	8/70 (11.4)	6/78 (7.7)	0.44
	Pre-stroke mRs >1	34/71 (47.9)	12/78 (15.4)	<0.001
	Current stroke event
	Admission Systolic BP, mmHg, mean (SD)	153 (22)	156 (18)	0.27
	Admission Diastolic BP, mmHg, mean (SD)	80 (13)	83 (13)	0.17
	Admission Glucose, mg/dl, mean (SD)	138 (40)	119 (34)	0.003
	Admission Platelet counts, mean (SD)	220 (86)	231 (63)	0.40
	Admission NIHSS, median (IQR)	18 (14 to 22)	8 (4 to 12)	<0.001
	Documented arterial occlusion
	None	11/71 (15.5)	35/80 (44.9)	<0.001
	ICA isolated or tandem with MCA	29/71 (40.8)	6/80 (7.7)
	MCA isolated	31/71 (43.7)	34/80 (43.6)
	Posterior circulation	0/71 (0.0)	3/80 (3.8)
	Cardioembolic etiology	39/71 (54.9)	33/78 (42.3)	0.12
	Treatment characteristics
	AIS treatment
	IV thrombolysis	19/71 (26.8)	49/80 (61.3)	<0.001
	Combined IV thrombolysis and Endovascular treatment (EVT)-	30/71 (42.3)	25/80 (31.3)
	EVT alone	22/71 (31.0)	6/80 (7.5)
	Mechanical endovascular treatment (MET)	36/71 (50.7)	18/80 (22.5)	<0.001
	Onset to treatment time, min, median (IQR)
	IV alone	154 (120 to 227)	152 (120 to 187)	0.78
	Combined IV-EVT	140 (115 to 160)	118 (90 to 145)	0.050
	EVT alone	309 (230 to 350)	325 (250 to 345)	0.48
	Values are number (%) unless otherwise as indicated. A favorable outcome is defined as 90-day mRS

Table 2 .

 2 Associations of biological markers with a favorable outcome.

		Favorable Outcome	Unadjusted	Adjusted 2
	Blood Biomakers, in ng/mL	No (n = 71)	Yes (n = 80)	p-Value 1	OR (95% CI)	p-Value 1
	Hemostasis
	uPA/urokinase	1.18 (1.04 to 1.34)	1.11 (0.93 to 1.31)	0.58	0.63 (0.41 to 0.97)	0.61
	Serpin E1/PAI-1	40 (20 to 67)	41 (21 to 66)	1.00	0.96 (0.60 to 1.55)	1.00
	Serpin C1/antithrombin-III	4672 (2963 to 6149)	4444 (2175 to 5615)	1.00	0.78 (0.48 to 1.26)	1.00
	Kallikrein 6/neurosin	4.7 (3.3 to 6.0)	4.8 (3.9 to 5.8)	1.00	1.00 (0.63 to 1.57)	1.00
	Alpha2 macroglobulin	2541 (1030)	2414 (865)	1.00	0.97 (0.61 to 1.55)	1.00
	Inflammation
	MPO	51 (37 to 74)	38 (28 to 50)	0.002	0.53 (0.31 to 0.89)	0.32
	CCL2/MCP-1	0.26 (0.18 to 0.31)	0.24 (0.19 to 0.32)	1.00	0.96 (0.63 to 1.44)	1.00
	Adiponectine	8689 (5118 to 14499)	7502 (5210 to 11821)	1.00	0.88 (0.54 to 1.41)	1.00
	Resistin	9.6 (7.0 to 12.5)	8.3 (6.6 to 10.6)	1.00	0.85 (0.54 to 1.34)	1.00
	cDNA	1.02 (0.93 to 1.13)	0.95 (0.86 to 1.04)	0.11	0.23 (0.08 to 0.66)	0.12
	CD40 Ligand	0.99 (0.63 to 1.93)	0.88 (0.64 to 1.29)	1.00	0.80 (0.50 to 1.28)	1.00
	Endothelial
	activation
	VCAM-1	1302 (834 to 1724)	838 (600 to 1215)	<0.001	0.53 (0.31 to 0.90)	0.32
	ICAM-1	301 (225 to 511)	258 (192 to 389)	1.00	0.67 (0.40 to 1.10)	1.00
	CD31/PECAM	9.7 (8.2 to 12.0)	9.7 (8.2 to 11.6)	1.00	1.01 (0.63 to 1.62)	1.00
	Tissue remodeling
	Total cathepsin S	6.1 (4.3 to 8.4)	4.9 (3.6 to 6.6)	0.093	0.60 (0.37 to 0.98)	0.72
	Osteopontin	40 (28 to 58)	26 (20 to 35)	0.001	0.34 (0.18 to 0.62)	0.008
	Cystain C	825 (711 to 1024)	725 (619 to 885)	0.068	0.61 (0.36 to 1.05)	1.00
	Neuropilin-1	229 (193 to 277)	196 (168 to 233)	0.011	0.64 (0.40 to 1.02)	0.98
	MMP-2	253 (192 to 295)	238 (188 to 282)	1.00	1.34 (0.82 to 2.19)	1.00
	MMP-3	8.6 (6.2 to 11.4)	9.1 (6.0 to 11.4)	1.00	0.95 (0.60 to 1.51)	1.00
	MMP-9	153 (77 to 321)	112 (61 to 201)	0.24	0.67 (0.42 to 1.05)	1.00
	MMP-13	0.33 (0.29 to 0.41)	0.37 (0.32 to 0.44)	1.00	0.86 (0.54 to 1.37)	1.00
	Values are median (IQR) except for Alpha2 macroglobulin, where mean (SD) is reported. For all biological markers
	(except Alpha2 macroglobulin), p-values and ORs were calculated on log-transformed values. ORs were calculated
	per one SD increase in biological markers. A favorable outcome is defined as 90-day mRS ≤ 2 or equal to pre-stroke
	mRS. 1 corrected for multiple comparisons using Holm Bonferroni method. 2 adjusted for age, admission NIHSS,
	documented occlusion (none vs. ICA occlusion vs. others arterial occlusion), and AIS treatments (including IV+-EVT
	approach). Abbreviations: AIS = acute ischemic stroke; CI = confidence interval; IV Intra-venous, EVT Endovascular
	treatment; ICA = internal carotid artery; IQR = interquartile range; IV = intravenous; mRS = modified Rankin Scale;
	OR = odds ratio; SD = standard deviation.

Table 3 .

 3 Associations of biological markers with a excellent outcome.

		Excellent Outcome	Unadjusted	Adjusted 2
	Blood Biomarkers, in ng/mL	No (n = 87)	Yes (n = 64)	p-Value 1	OR (95% CI)	p-Value 1
	Hemostasis
	uPA/urokinase	1.18 (1.02 to 1.34)	1.08 (0.90 to 1.29)	0.27	0.61 (0.38 to 0.98)	0.67
	Serpin E1/PAI-1	39 (19 to 65)	42 (22 to 67)	1.00	1.14 (0.74 to 1.75)	1.00
	Serpin C1/antithrombin-III	4481 (2653 to 5581)	4567 (2951 to 5870)	1.00	1.12 (0.74 to 1.71)	1.00
	Kallikrein 6/neurosin	4.6 (3.5 to 5.7)	4.9 (4.1 to 5.9)	1.00	1.19 (0.77 to 1.81)	1.00
	Alpha2 macroglobulin	2459 (986)	2494 (805)	1.00	1.23 (0.81 to 1.86)	1.00
	Inflammation
	MPO	49 (35 to 71)	37 (27 to 50)	0.009	0.60 (0.37 to 0.97)	0.62
	CCL2/MCP-1	0.25 (0.18 to 0.32)	0.23 (0.19 to 0.30)	1.00	0.92 (0.62 to 1.37)	1.00
	Adiponectine	8100 (4226 to 14499)	7619 (5363 to 11781)	1.00	1.05 (0.68 to 1.61)	1.00
	Resistin	9.1 (6.7 to 11.8)	8.3 (6.6 to 10.7)	1.00	1.08 (0.72 to 1.61)	1.00
	cDNA	1.02 (0.93 to 1.12)	0.95 (0.85 to 1.03)	1.00	0.76 (0.49 to 1.17)	1.00
	CD40 Ligand	0.91 (0.63 to 1.93)	0.89 (0.64 to 1.24)	0.37	0.65 (0.41 to 1.02)	0.91
	Endothelial
	activation
	VCAM-1	1237 (826 to 1682)	821 (592 to 1040)	<0.001	0.54 (0.33 to 0.89)	0.29
	ICAM-1	309 (228 to 530)	228 (177 to 337)	0.27	0.59 (0.37 to 0.92)	0.37
	CD31/PECAM	9.9 (8.3 to 12.1)	9.5 (8.0 to 11.1)	1.00	0.74 (0.47 to 1.15)	1.00
	Tissue remodeling
	Total cathepsin S	5.9 (4.3 to 8.2)	4.5 (3.5 to 6.5)	0.048	0.58 (0.37 to 0.91)	0.36
	Osteopontin	38 (24 to 55)	26 (20 to 33)	0.010	0.49 (0.29 to 0.81)	0.12
	Cystain C	812 (694 to 1014)	753 (604 to 938)	0.68	0.89 (0.55 to 1.44)	1.00
	Neuropilin-1	226 (188 to 274)	191 (167 to 233)	0.048	0.71 (0.45 to 1.09)	1.00
	MMP-2	252 (193 to 295)	235 (181 to 279)	1.00	1.19 (0.74 to 1.89)	1.00
	MMP-3	8.6 (6.1 to 11.4)	9.1 (6.5 to 11.4)	1.00	1.03 (0.67 to 1.56)	1.00
	MMP-9	138 (67 to 264)	120 (63 to 209)	1.00	0.90 (0.60 to 1.34)	1.00
	MMP-13	0.34 (0.29 to 0.42)	0.37 (0.29 to 0.42)	1.00	0.83 (0.52 to 1.32)	1.00

Table 4 .

 4 Associations of biological markers with early neurological improvement.

		Early Neurological Improvement	Unadjusted	Adjusted 2
	Blood Biomarkers, in ng/mL	No (n = 79)	Yes (n = 72)	p-Value 1	OR (95% CI)	p-Value 1
	Hemostasis
	uPA/urokinase	1.15 (1.01 to 1.36)	1.15 (0.95 to 1.29)	1.00	0.98 (0.67 to 1.41)	1.00
	Serpin E1/PAI-1	40 (23 to 67)	41 (20 to 66)	1.00	0.84 (0.57 to 1.22)	1.00
	Serpin C1/antithrombin-III	4693 (2930 to 6055)	4314 (2881 to 5639)	1.00	0.83 (0.56 to 1.23)	1.00
	Kallikrein 6/neurosin	4.8 (3.6 to 5.8)	4.8 (3.8 to 6.0)	1.00	0.87 (0.58 to 1.29)	1.00
	Alpha2 macroglobulin	2571 (1036)	2367 (829)	1.00	0.80 (0.54 to 1.17)	1.00
	Inflammation
	MPO	49 (37 to 73)	37 (27 to 52)	0.005	0.51 (0.32 to 0.80)	0.064
	CCL2/MCP-1	0.22 (0.16 to 0.31)	0.25 (0.20 to 0.31)	1.00	1.11 (0.77 to 1.59)	1.00
	Adiponectine	8373 (5273 to 14587)	7454 (5051 to 11788)	1.00	0.86 (0.58 to 1.25)	1.00
	Resistin	9.2 (7.0 to 12.5)	7.7 (6.4 to 10.6)	0.83	0.70 (0.47 to 1.05)	1.00
	cDNA	0.98 (0.92 to 1.10)	0.96 (0.88 to 1.08)	1.00	1.23 (0.83 to 1.81)	1.00
	CD40 Ligand	0.98 (0.65 to 1.65)	0.86 (0.60 to 1.38)	1.00	0.82 (0.56 to 1.20)	1.00
	Endothelial
	activation
	VCAM-1	1034 (730 to 1641)	908 (663 to 1405)	1.00	0.98 (0.66 to 1.46)	1.00
	ICAM-1	309 (225 to 571)	255 (192 to 337)	1.00	0.76 (0.52 to 1.09)	1.00
	CD31/PECAM	9.7 (8.3 to 11.9)	9.8 (8.0 to 11.6)	1.00	0.87 (0.60 to 1.27)	1.00
	Tissue remodeling
	Total cathepsin S	6.0 (4.4 to 8.4)	4.4 (3.6 to 6.5)	0.007	0.54 (0.35 to 0.81)	0.064
	Osteopontin	32 (23 to 52)	30 (20 to 45)	1.00	0.80 (0.55 to 1.17)	1.00
	Cystain C	812 (666 to 958)	758 (651 to 975)	1.00	0.89 (0.57 to 1.36)	1.00
	Neuropilin-1	227 (188 to 268)	203 (170 to 236)	0.55	0.76 (0.52 to 1.11)	1.00
	MMP-2	248 (192 to 282)	242 (192 to 292)	1.00	1.30 (0.86 to 1.97)	1.00
	MMP-3	8.1 (5.8 to 11.3)	9.3 (7.1 to 12.4)	0.43	1.60 (1.09 to 2.34)	0.29
	MMP-9	159 (85 to 290)	97 (57 to 201)	0.059	0.62 (0.42 to 0.90)	0.23
	MMP-13	0.34 (0.29 to 0.41)	0.38 (0.31 to 0.44)	1.00	1.03 (0.69 to 1.53)	1.00

J. Clin. Med. 2020, 9, 4028; doi:10.3390/jcm9124028 www.mdpi.com/journal/jcm

J. Clin. Med. 2020, 9, x FOR PEER REVIEW

Funding: This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), by a non-profit grant from the MSDAvenir fund, and by SOS-ATTAQUE CEREBRALE Association (a not-for-profit stroke survivor organization). Grant from fondation pour la recherche sur les AVC numero de dossier AVC20180600362.

Supplementary Materials:

The following are available online at http://www.mdpi.com/2077-0383/9/12/4028/s1, Table S1: Patient's and treatment characteristics in the included and non-included patients. Table S2: Distribution of biological markers in the included patients.

Conflicts of Interest:

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.


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Remote sensing of the Earth's soil color in space and time

Rodnei Rizzo, Alexandre M.

  J.-C Wadoux, José A.M. Demattê, Budiman Minasny, Vidal Barrón, Eyal Ben-Dor, Nicolas Francos, Igor Savin, Raul Poppiel, Nelida E.Q. Silvero, et al. To cite this version: Rodnei Rizzo, Alexandre M.J.-C Wadoux, José A.M. Demattê, Budiman Minasny, Vidal Barrón, et al.. Remote sensing of the Earth's soil color in space and time. Remote Sensing of Environment, 2023, 299, pp.113845. 10.1016/j.rse.2023.113845. hal-04346311


Lukas Puts
email: l.puts@tue.nl

Xaveer Leijtens

Pavel Cheben

Jens Schmid

Sander Reniers

AND Daniele Melati

Anti-reflection subwavelength gratings for InP-based waveguide facets

We demonstrate the anti-reflection properties of lithographically defined subwavelength gratings applied to the facets of InP-based integrated waveguides. The subwavelength gratings are based on the gradient index effect to create a smooth index transition between the core material and air, achieving reflections below -30 dB at a wavelength of 1550 nm for both TE and TM polarized modes, as shown by 3D finite-difference timedomain simulations. Characterizations performed on the experimental InP membrane-on-silicon platform using Mach-Zehnder interferometers test structures are in excellent agreement with simulation predictions.

Controlling reflections at waveguide facets is crucial for optimal performance of photonic integrated devices and circuits. A notable example is the strong effect that small reflections can have on the dynamics of integrated lasers [1,2]. For fiber-tochip edge couplers low reflections are needed to achieve a high coupling efficiency. Waveguide facets represent one of the main sources of reflections, generated by the abrupt index transitions between the waveguide materials and air. For typical single mode waveguides in silicon and III-V semiconductor platforms, cleaved facets without adaptation structures have a strong reflectivity of about 30% [3]. Commonly, single or multilayer anti-reflection (AR) thin films with a precisely engineered thickness and refractive index are used to reduce these reflections. However, this comes with some important drawbacks. Complex vacuum deposition techniques are required to apply films on a cleaved facet, which can be done only as a post-processing step, increasing fabrication complexity. Furthermore, the films may become mechanically unstable under thermal cycling, resulting in loss of performance [4]. Another approach that is often applied is the use of angled facets. By changing the facet angle, reflections of about -40 dB for the fundamental mode has been demonstrated in III-V waveguides [5]. However, by increasing the facet angle, higher order mode reflections can be generated. This can be especially problematic in wide waveguides support-ing higher order modes [6].

Another strategy to minimize reflections is the patterning of the waveguide facet with a subwavelength grating (SWG). This type of structure has been used in a wide range of applications to control reflections. For example, SWGs have been applied on bulk surfaces such as solar cells to optimize performance [7]. The same concept has been exploited to reduce reflections from the facets of quantum cascade lasers by patterning the facets using focused ion beam (FIB) milling after chip fabrication. Reflections as low as 1-3% have been achieved using this method [8]. However, FIB milling can only be applied during post-processing. Moreover, ion sputtering and a diverging ion beam resulted in significant differences between the designed and the fabricated grating geometry, indicating FIB might not be ideal to fabricate SWGs. The use of lithographically defined SWGs as anti-reflective coatings, first demonstrated by Schmid et al. [4], eliminates the need for chip-level post-processing. A grating with triangular teeth was fabricated using electron beam lithography (EBL) on a Silicon-on-Insulator (SOI) platform. Reflections as low as 2% and 2.4% for the fundamental TE and TM polarized modes were achieved, respectively [9]. While extensively studied for SOI waveguides [10][11][12][13], little investigations have been reported so far on the use of SWGs in InP-based platforms. The possibility to control reflections by subwavelength patterned geometries is of particular interest because of the wide range of active components available in III-V platforms, which often heavily rely on thin-film AR facet coatings.

In this paper, we simulate, design and experimentally demonstrate an AR subwavelength waveguide facet grating on the InP membrane on silicon (IMOS) platform [14]. The IMOS platform comprises a 300 nm thick InP photonic membrane on a standard silicon wafer. The layer stack for a standard 400 nm wide single-mode IMOS waveguide is shown in Fig. 1(a). First, the InP waveguide pattern is transferred to a small InP wafer using EBL. The wafer is then flipped and bonded to a silicon wafer using the adhesive polymer divinylsiloxane-bis-benzocyclobutene (DVS-BCB) and a 200 nm thick SiO 2 bonding layer. The InP substrate and InGaAs etch-stop layers are removed by selective wet-chemical etching. The thin patterned InP membrane remains on the silicon wafer [14]. Due to the large index difference between InP (n=3.17), BCB (n=1.54), and SiO 2 (n=1.45), very compact waveguides can be realized. The resulting electric field distribution for the fundamental TE and TM modes is shown in Fig. 1(c) and (d). Most of the electric field of the fundamental TE mode is concentrated in the waveguide core, whereas the TM mode is primarily localized in the oxide cladding below and air above the core. To realize the AR structure, the waveguide core is patterned with a gradient index (GRIN) SWG consisting of an array of triangles, as depicted in Fig. 1(b). When the period of the grating is smaller than the wavelength in the medium, the structure can be approximated as an equivalent material with a refractive index gradually changing from that of the waveguide core to the index of the cladding [15]. The gradual transition from core to cladding suppresses facets reflections by eliminating the abrupt termination of the waveguide. Since index grading does not rely on interference effects, it is inherently less wavelength sensitive compared to common thin film AR layers [4]. A combination of 3D finite-difference time-domain (FDTD) simulations for accurate reflection evaluation and 2D FDTD for tolerance analysis was used to design the AR structures. The model comprises a waveguide and a triangular grating with a fixed subwavelength period of 400 nm, as shown in Fig. 1(b). Grating length is varied from 0 to 2 µm, and the waveguide width is varied from 2 to 4 µm. The fundamental TE or TM mode is injected from the left at λ 0 = 1550 nm towards the grating. A power monitor left to the source measures the amount of light reflected from the SWG structure.

The TE and TM reflections calculated by 3D FDTD as a function of the grating teeth length are shown in Fig. 2 and3, respectively. For clarity, the simulation results for a 2 µm wide SWG are marked with the blue dots, the unmarked curves show the results for a 4 µm wide SWG. The dash-dotted lines show the total reflections (TE), the solid lines show the modal reflections (TE 0 ). The latter refers to the amount of reflected power coupled into the counter propagating fundamental mode. At a grating teeth length of 0 nm the structure is simply a flat facet with a reflection of ∼25%, as expected (Fig. 2). From these simulations it is clear that the facet reflection decreases rapidly with increasing grating teeth length. The TE 0 reflection drops to about -30 dB for a grating teeth length of approximately 0.5 µm. The difference between the modal and total reflection simulations is small, indicating that mode conversion is small. The oscillatory behavior of the reflection results in three other minima at 1 µm, 1.4 µm and 1.85 µm. Simulated reflections for these minima are between -30 and -35 dB. The width of the waveguide has only a small influence on the reflection. From these simulations, the preferred design spot for a substantial reduction in TE reflection is the first minimum, at about 0.51 µm. This results in a compact device with the smallest possible aspect ratio. Even lower reflection could be achieved for longer grating teeth lengths, that however would require structures with a larger aspect ratio, making fabrication more challenging.

The results for TM simulations are shown in Fig. 3. The simulated structures are the same as described in the previous paragraph. Without any grating teeth (length of 0 nm) the reflections are around 30%, which decrease to values below -30 dB for grating teeth length larger than 1.4 µm. The SWG teeth are parallel to the oscillations of the electric field for the TM polarization and as a result the anti-reflection effect is less strong compared to TE polarization. This was previously observed also in AR SWGs on SOI waveguides [4]. The preferred design spot in this case is 1.55 µm, at which a reflection of about -35 dB is achieved.

Next, fabrication tolerances of the proposed designs are investigated. Due to the diffraction limit and electron backscattering in the photoresist, the resolution for EBL is limited to approximately 20 nm. There are two regions where the SWG feature size is below 20 nm and are hence difficult to define using EBL. The tip of each tooth will result in a rounded shape with a certain radius and the region between the bases of two adjacent teeth will not be defined accurately. Additionally, for an SWG with high aspect ratios, latter regions can be also difficult to etch, due to the etch lag effect [16]. In order to ensure a reliable fabrication, a 'stub' is placed between the bases of adjacent teeth, as shown in Fig. 1 (b). The size of this stub is made equal to the resolution of the EBL, decreasing the etch lag effect in this particular region. 2D FDTD simulations show that a stub of 20 nm adds approximately 3.2 and 2.1 dB to the total and modal TE reflections, respectively. Random and systematic process-induced fluctuations such as variations in focus, dose, and resist quality influence critical dimensions (CD). These fluctuations may result in uniform CD variations of several nanometers with respect to In order to use this structure to measure the SWG reflectivity, the output power of the bar (P 3 ) and cross (P 4 ) ports as a function of the wavelength can be derived using the expression for the transfer function of a single MZI [18]. The contrast ratio C, defined as the ratio between the minima and maxima of the fringes in the MZI transmission spectrum for either of the two output ports P 3 and P 4 , can be calculated as follows:

C = P out, max P out, min = √ R 1 + √ R 2 α m e -αl + 2 2 √ R 1 + √ R 2 α m e -αl -2 2 (1)
where R 1 and R 2 are the corresponding SWG reflectivities, α m the MMI coupler loss, α the waveguide propagation loss, and l the MZI arm length difference. Assuming R 1 = R 2 = R, the reflectivity R can hence be determined from the measured contrast ratio. A total of 20 MZIs with 4 µm wide SWGs and grating teeth length ranging from 0 to 2 µm were designed on a 4×6 mm 2 IMOS cell. The grating period was fixed at 400 nm. Two identical cells were patterned by EBL and proximity effect correction (PEC) and shape correction were applied to the GDS file to compensate for electron back scattering and optimize the dose locally. TE polarized light was coupled in and out of the circuit using surface grating couplers optimized for TE coupling (Fig. 5). Due to variations in the fabrication, the optimal coupling efficiency of the surface grating couplers was shifted to 1535 nm from the designed 1550 nm. The wavelength of the input light was swept from 1465 to 1575 nm using a tuneable laser source. The chip temperature was maintained at 21°C using a Peltier element mounted on a copper chuck. The output light intensity was measured by a power meter. Two examples of the measured spectra at the output port P 3 for the test MZI with SWGs teeth lengths of L=200 and L=500 nm are shown in Fig. 6. The latter is the preferred design spot for the TE polarized mode and, as expected, its anti-reflection properties translate into a reduced contrast ratio of the spectral fringes compared to the SWG with a grating teeth length of 200 nm.

The mean and standard deviation of the contrast ratio C for each of the 20 MZIs of one design cell was determined from multiple fringes in the spectral range from 1520 to 1540 nm of the individual devices. The calculated relative reflections derived from Eq. ( 1) are then normalized using the 3D FDTD simulations of the SWG with a grating teeth length of 200 nm. The solid line in Fig. 7 shows the results of this analysis as a function of the grating teeth length. Squares mark the mean relative reflections and the error bars the standard deviations. The dashed line shows the modal TE 3D FDTD simulation results for reference (same data as in Fig. 2). Results of the simulations and measurements are in a very good agreement and show a similar trend of reflection as function of grating teeth length. The reflections drop rapidly with increasing grating teeth length and similar to the simulations the first minimum is located approximately at 500 nm at a value of -18 dB below the expected flat facet reflec- tion of -6.8 dB. Relative reflectivity between 600 and 1600 nm is substantially higher compared to the simulations. This is likely related to the challenges in etching SWGs with large aspect ratios. Moreover, the reflection of a flat facet is substantially lower than what is expected from simulations. A possible explanation for this is that non-perfect vertical etching may have resulted in a slightly slanted facet which would suppress coupling of the reflected light to the waveguide mode.

In conclusion, we have designed and experimentally demonstrated an effective anti-reflection strategy for InP IMOS waveguides by exploiting subwavelength gratings. These lithographically defined structures are particularly interesting for InP-based technology platforms since the performance of active components often relies heavily on anti-reflection coatings deposited during post-processing. Simulated TE 0 mode reflections are as low as -30 dB for an SWG with a grating teeth length of 0.51 µm, width of 4 µm and a fixed period of 400 nm. This results in a compact design with a relatively low aspect ratio, which is beneficial for fabrication. Reflections as low as -35 dB are achieved for the TM 0 mode for SWGs with a grating teeth length of 1.55 µm. The anti-reflection effect was experimentally demonstrated on waveguide facets using Mach-Zehnder interferometers as test structures. Reflections for a 4 µm wide SWG and grating teeth lengths varied from 0 to 2 µm yield excellent agreement with 3D FDTD simulations.

Fig. 1 .

 1 Fig. 1. (a) Schematic cross-section of a standard IMOS waveguide, indicating the materials with respective refractive indices and dimensions. (b) Top view of the SWG patterned on the waveguide facet and FDTD simulation layout. (c) and (d) Fundamental TE and TM electric field profiles in a 400 nm wide IMOS waveguide, respectively.

Fig. 2 .

 2 Fig. 2. 3D FDTD simulation results for the reflection of the TE polarized mode as a function of the grating teeth length.

Fig. 3 . 2 Fig. 4 .

 324 Fig. 3. 3D FDTD simulation of the reflection of the TM polarized mode as a function of the grating teeth length.

Fig. 5 .

 5 Fig. 5. Photograph of part of the IMOS chip showing three Mach-Zehnder interferometers and the input and output fibers at an angle of 10°for optimal coupling.

Fig. 7 .

 7 Fig. 7. Overlay of 3D FDTD simulations and experimental results in the IMOS platform. The error bars show the standard deviation in the measurement.

  Output power at P 3 of two MZIs with facet gratings lengths of 200 nm and 500 nm. Lower fringe contrast of the SWG with longer grating teeth length is due to the AR properties. Contrast ratio for these test structures is 2.484±0.332 and 1.176±0.0815 for lengths of 200 and 500 nm, respectively. To prevent cluttering of data points, only a quarter of the measurements are marked.

		-14
		-15
		-16
	Measured power [dBm]	-21 -20 -19 -18 -17				L=200 L=500
		-22
		-23
		-24
		1530	1532	1534	1536	1538	1540
				Wavelength [nm]
	Fig. 6.

Acknowledgments. The authors would like to thank Marc Spiegelberg and Jeroen Bolk for their contribution to the design and characterization. Nazca Design was used to generate the mask layout in this work.

Disclosures.

The authors declare no conflicts of interest.