Study design

All stroke experiments were performed in accordance with the recently published ARRIVE guidelines (http://www.nc3rs.org/ARRIVE). An independent person not involved in data acquisition and analysis randomly assigned animals to the operators. We performed surgery and evaluation of all read-out parameters while being blinded to the experimental groups. Animals were excluded from end-point analyses (exclusion criteria) if death occurred within 24 h after MCAO, if subarachnoidal hemorrhage (SAH) or intracerebral hemorrhage (ICH) occurred (as macroscopically assessed during brain sampling), or if, 60 min after tMCAO, Bederson scores were = 0. Of the 242 mice subjected to MCAO, 38 mice (15.7%) met at least one of these exclusion criteria after randomization and were withdrawn from the study resulting in 204 included mice. The drop-out rates was 22 mice in the vehicle group vs. 16 mice in the BAY60-2770 treated group. The study design was defined based on a power animal calculation analysis49 for all groups involved in the study considering different compounds (BAY58-2667 and BAY60-2770), doses (30 µg/kg, 10 µg/kg), treatment time-points (1 h and 4 h post-stroke), age of the animals (adults and middle-aged) based on mean infarct size, standard deviation a and number of animals used always comparing vehicle and treatment administration (Table S3). The study was conducted in accordance with institutional guidelines (University of Würzburg, Germany) for the use of experimental animals, and the protocols were approved by governmental authorities (Regierung von Unterfranken, Würzburg, Germany).

Human interactome analysis

We used an integrated human interactome containing 141,150 experimentally documented physical interactions between 13,329 proteins, as curated by ref. 3 From the same study, we used 299 diseases defined by MeSH classification and their associated genes that were curated from OMIM and GWAS databases to identify disease modules within the interactome. To remove potential redundancy among diseases, we calculated the Jaccard Index between every pair of diseases using the genes associated to them and filtered the disease terms that had a Jaccard Index higher than 0.5 to a disease in the data set, yielding 132 diseases. For each of the sGC proteins, guanylate cyclase soluble subunit beta-1 (GUCY1B3), guanylate cyclase soluble subunit alpha-2 (GUCY1A2) and subunit alpha-3 (GUCY1A3), we calculated the minimum shortest path length to known disease genes for each of the 132 diseases. As control, we further computed the minimum shortest path lengths from the sGCα proteins to 1000 randomly chosen genes sets containing the same number of genes as the respective disease. From the mean and standard deviation of these random controls we then calculated a z-score, z = (d observed −<d random >)/σ random , to quantify the significance of the closeness of the sGCα proteins to a given disease. We converted z-scores to P-values and then adjusted the P-values using the Benjamini–Hochberg multiple hypothesis testing correction method. We set the alpha value for FDR to 25% and considered any disease with an FDR less than or equal to this value as significantly close to the sGCα protein.

Disease–disease relationships

The four different relationships among the diseases in the cluster depicted in Fig. 1c–f were obtained as follows: (i) We determined the number of shared genes (Fig. 1c) from gene-disease association data combined from ref. 3 to ref. 50 (ii) We computed the number of physical protein interactions connecting the gene products of the two respective diseases using interactome data from.3 For every disease pair, we first checked the number of interactions connecting the genes from one disease to the genes in the other disease (see the Supplemenary Material for the pseudocode for this procedure). We then compared the number of observed interactions between disease genes to the number of interactions one would observe between randomly selected gene pairs and used the normalized score (z-score) to decide whether the disease genes were more connected to each other compared to random pairs of genes. The expected distribution of the number of interacting gene pairs was generated using 1000 randomly chosen pairs of gene sets containing the same number of genes as the two diseases in consideration. The z-score was calculated using the mean and standard deviation of this distribution. Links in Fig. 1d indicate a significant number of interacting proteins. (iii) We extracted disease pairs with high symptom similarity4 (Fig. 1e). Only the symptoms that had strong support (TF-IDF score > 3.5) in the original data set were considered in the analysis. (iv) Elevated co-morbidities calculated from disease pairs reported in insurance claims (Fig. 1f) were taken from ref. 51 The ICD9 codes provided in the original data set were converted to MeSH identifiers using the mapping provided by Disease Ontology and only the diseases that could be unambiguously mapped were included in the analysis. The pairwise disease–disease relationship scores corresponding to the calculated gene and interaction sharing, symptom similarity and comorbidity are available in Tables S2A-D.

Centrality analysis of diseases in diseasomes

To prioritize the diseases, we generated four diseasomes based on the disease–disease relationship scores above. We kept only the links that correspond to non-spurious relationships between diseases by including disease pairs that shared at least one gene (n > 0); had positive shared protein interaction score (z > 0); showed symptom similarity (Jaccard index > 0.5); and are known to be comorbid (relative risk > 1) in the four diseasomes, respectively. We then calculated degree centrality of each disease within these diseasomes and averaged the centrality values as the final centrality value of the disease across diseasomes (Table 1). To ensure that the incompleteness of the interaction data did not have a significant effect on the ranking of the diseases, we also checked the centrality of the diseases without using the interactome-based diseasome and found that stroke remained as the most central disease across the diseasomes (Table S4). The coverage and average degree of the individual diseasomes are given in Table S1.

sGC activity

The determination of sGC activity was performed in homogenates of mouse stroked ipsilateral cortex and basal ganglia vs. non-stroke cortex and basal ganglia, as previously described.23 Briefly, crude brain homogenates of stroked and non-stroked C57Bl/6 mice were measured as the formation of cGMP at 37 C during 10 min in a total incubation volume of 100 μl containing 50 mM triethanolamine–HCl (pH 7.4, Sigma), 3 mM MgCl, 3 mM glutathione (Carl Roth), 1 mM 3-isobutyl-1-methylxanthine (IBMX, Enzo LifeSciences), 100 mM zaprinast (Enzo LifeSciences), 5 mM creatine phosphate (CalBiochem), 0.25 mg/ml creatine kinase (CalBiochem), and 500 mM GTP. The reaction was started by simultaneous addition of the crude brain homogenates and either DEA/NO (Enzo LifeSciences) or BAY58-2667 (Adipogene), respectively. After incubation of each sample (n = 3 each per group) for 10 min the reaction was stopped by boiling for 10 min at 95 °C. Thereafter, the amount of cGMP was subsequently determined by an enzyme immunoassay (ENZO cGMP EIA kit) using different sample dilutions in the linear range.

In vivo MCAO ischemia model

C57BL/6 mice were subjected to middle cerebral artery occlusion (MCAO) followed by 24 h of reperfusion. Focal cerebral ischemia was induced by 60 min transient middle cerebral artery occlusion (tMCAO) as described in refs. 52,53,54 Mice were anesthetized with 2.5% isoflurane (Abbott) in a 70% N 2 O/30% O 2 mixture. Core body temperature was maintained at 37 °C throughout surgery by using a feedback-controlled heating device. Following a midline skin incision in the neck, the proximal common carotid artery and the external carotid artery were ligated and a standardized silicon rubber-coated 6.0 nylon monofilament (6021; Doccol) was inserted and advanced via the right internal carotid artery to occlude the origin of the right MCA. The intraluminal suture was left in situ for 60 min. Then animals were re-anesthetized and the occluding monofilament was withdrawn to allow for reperfusion. For permanent MCAO (pMCAO) the occluding filament was left in situ until sacrificing the animals.54 Operation time per animal did not exceed 10 min.

Treatment with apo-sGC activators

BAY60-2270 and BAY 58-2667 were dissolved in a mixture of Transcutol/Cremophor/water in a ratio of 10/20/70.55 BAY 58-2667 (30 µg/kg), BAY60-2270 (5 µg/kg or 10 µg/kg) or vehicle (Transcutol/Cremophor/water in a ratio of 10/20/70) was injected i.v. at the time of reperfusion (immediately after removal of the filament) except in one group where BAY 60-2270 was injected 3 h after removal of the filament that means 4 h after induction of tMCAO.

Determination of blood-brain-barrier leakage and brain edema

To determine blood–brain–barrier leakage 100 µl of 2% Evan’s Blue tracer (Sigma Aldrich) diluted in 0.9% NaCl was i.v. injected 1 h after the induction of tMCAO. After 24 h mice were sacrificed and brains were quickly removed and cut in 2 mm thick coronal sections using a mouse brain slice matrix (Harvard Apparatus). Brain slices were fixed in 4% PFA at 4 °C for 2 h in the dark. Then, brain slices were cut into small pieces using a scalpel and then transferred into Eppendorf tubes. 500 µl Formamid was added to each tube and incubated for 24 h at 50 °C in the dark. Tubes were centrifuged for 20 min at 16,000 g and 50 µl of the supernatant was transferred to a 96 well plate. Fluorescence intensity was determined in duplicates by a microplate fluorescence reader (Fluorosan Ascent, Thermo Scientific) with an excitation at 620 nm and emission at 680 nm. The concentration for each sample was calculated from a standard curve using linear regression analysis.

Invasive hemodynamics

For the assessment of blood pressure mice were anesthetized with 2.0% isoflurane and catheterized via the right carotid artery with a high-fidelity 1.4 F Millar microtip catheter (Milar Instruments), as described in ref. 56 Hemodynamic data were digitized via a MacLab system (AD Instruments) connected to an Apple G4 PowerPC computer and analyzed.

Laser Doppler flowmetry

Laser Doppler flowmetry (Moor Instruments) was used to monitor relative regional cerebral blood flow (rCBF) in the right MCA area.57 For this procedure, a small incision was made in the skin overlying the temporal muscle, and a 0.7 mm flexible Laser Doppler probe (model P10) was positioned perpendicular to the superior portion of the temporal bone (6 mm lateral and 2 mm posterior from bregma). This position corresponds to the core of the ischemic territory. rCBF was measured serially at baseline (before ischemia), immediately after insertion of the occluding filament (ischemia), immediately after removal of the occluding filament (reperfusion) and again 1 h, 2 h, and 3 h after reperfusion.

Determination of infarct size

After sacrificing the mice, brains were quickly removed and cut in three 2-mm thick coronal sections using a mouse brain slice matrix (Harvard Apparatus). The slices were stained for 10 min at 37 °C with 2% 2,3,5-triphenyltetrazolium chloride (TTC; Sigma-Aldrich) in PBS to visualize the infarctions.58 Indirect, i.e., corrected for brain edema, infarct volumes were calculated by volumetry (ImageJ software, National Institutes of Health, USA) according to the following equation: V indirect (mm3) = V infarct ×(1−(V ih −V ch )/V ch ), where the term (V ih −V ch ) represents the volume difference between the ischemic hemisphere and the control hemisphere and (V ih −V ch )/V ch expresses this difference as a percentage of the control hemisphere.

Assessment of neuro-functional outcomes

Three different neuro-motor functioning tests were assessed in all mice groups (male, female, middle-aged) treated 1 h post-ischemia. For the grip test59 the mouse was placed midway on a string between two supports and rated as follows: 0, falls off; 1, hangs on to string by one or both fore paws; 2, as for 1, and attempts to climb on to string; 3, hangs on to string by one or both fore paws plus one or both hind paws; 4, hangs on to string by fore and hind paws plus tail wrapped around string; 5, escape (to the supports). For the elevated body swing test the mice was held ~1 cm from the base of its tail. Then, it was elevated above the surface in the vertical axis. A swing was considered whenever the animal moved its head out of the vertical axis to either the left or the right side (> 10°). To evaluate limb strength, the four-paw wire hanging test was performed. The mouse was placed on the center of the wire with a diameter of 8 cm and then the wire was slowly inverted and placed at 40 cm above a paper towel bedding. The time until the mouse fell from the wire was recorded, and the maximum time was set to 120 s.

Staining of activated microglia/macrophages

Cryo-embedded slices were fixed in 4% PFA in PBS. Blocking of epitopes was achieved by pre-treatment with 5% bovine serum albumin (BSA) in PBS for 45 min to prevent unspecific binding. Rat anti-mouse CD11b (microglia/macrophages; MCA711, AbD Serotec) at a dilution of 1:100 in PBS containing 1% BSA was added overnight at 4 °C. Afterwards, slides were incubated with a biotinylated anti-rat IgG (BA-4001, Vector Laboratories) diluted 1:100 in PBS containing 1% BSA for 45 min at room temperature. Following treatment with Avidin/Biotin blocking solution (Avidin/Biotin Blocking Kit, Sp-2001, Vector Laboratories) to inhibit endogenous peroxidase activity, the secondary antibody was linked via streptavidin to a biotinylated peroxidase (POD) according to the manufacturer’s instructions (Vectorstain ABC Kit, Peroxidase Standard PK-4000, Vector Laboratories). Antigens were visualized via POD using the chromogen 3,3′- Diaminobenzidin (DAB) (Kem-En-Tec Diagnostics). For quantification of immune cells identical brain sections (thickness 10 µm) at the level of the basal ganglia (0.5 mm anterior from bregma) were selected and cell counting was performed from 5 subsequent slices (distance 100 µm) from 4 different animals.

Apoptosis measurement

Apoptotic neurons in the ischemic hemisphere 24 h after tMCAO were visualized by TUNEL on cryo-embedded slices. Brain slices (10 µm thickness) were fixed in acetone for 10 min and blocked for 1 h in 5% BSA in PBS containing 1% Goat Serum and 0.3% Triton to prevent nonspecific binding. A mouse antibody to NeuN (MAB377, Millipore, 1:1000 in PBS) was applied over night at 4 °C. Proteins were detected by 45 min of incubation with Dylight 488–conjugated goat antibody to mouse secondary antibodies (polyclonal, ab96871, Abcam) at a dilution of 1:200 in 1% BSA in PBS. TUNEL positive cells were stained using the in situ cell death detection kit TMR red (Roche 12 156 792 910) following the manual instructions. Negative controls included omission of primary or secondary antibody and gave no signals (not shown).

Western blot analysis

Brains were extracted from sacrificed stroke animals and classified as ipsilateral and contralateral. These were fast-frozen in liquid nitrogen, then stored at −80 °C until the time of analysis. Snap-frozen brains were crunched in liquid nitrogen and the brain powder was transferred into an eppendorf tube. Then hot Laemmli buffer (Bio-Rad, Veenendaal, The Netherlands), pre-heated at 95 °C and containing 5% β-mercaptoethanol, was added and the powder was lysed and denaturated for 10 min at 65 °C. Ultrasounds (Hielscher, Teltow, Germany) were used to homogenize the samples and then they were centrifuged at 12,000×g for 15 min at 4 °C. The supernatant was collected and stored at −80 °C. The total protein concentration was measured in the tissue supernatant by using the RC DC assay (Bio-Rad, Veenendaal, The Netherlands). Brain lysates were separated on a NuPAGE Novex 10% Bis–Tris Midi Gel 1.0 mm × 26 well, (Novex Life Technologies, Bleiswijk, The Netherlands) with the XCell SureLock Midi-Cell running tank (Life Technologies, Bleiswijk, The Netherlands). According to the invitrogen protocol MES running buffer was used (Life Technologies, Bleiswijk, The Netherlands).15 µg protein per well were loaded and laemmli-buffer containing plus 5% β-mercaptoethanol was used to adjust the volume and 0.3 µl protein marker were applied (LI-COR IRDye protein molecular weight marker, Licor Westburg, Leusden, The Netherlands). Proteins were transferred to nitrocellulose membranes using NuPAGE Iblot Gel Transfer Stacks Nitrocellulose (Novex Life Technologies, Bleiswijk, The Netherlands) on the iBlot Gel Transfer Device (Life Technologies, Bleiswijk, The Netherlands). The blot was stained with Ponceau S (Sigma Aldrich, Zwijndrecht, The Netherlands) to check for complete protein transfer. Non-specific reactivity was blocked by incubating the membranes for 1 h at room temperature with blocking buffer, 1 part Odyssey buffer (Licor Westburg, Leusden, The Netherlands) and 1 part PBS (Merck Chemicals, Amsterdam, The Netherlands). Hybridization also took place in Odyssey-PBS Blocking Buffer. The membranes were incubated with primary antibody overnight at 4 °C with a self-made polyclonal rabbit sGC-α1 antibody (dilution 1:2000) or with a polyclonal rabbit sGC-β1 antibody (dilution 1:2000). After three washing steps with PBS containing 0.1% Tween-20 (Merck Chemicals, Amsterdam, The Netherlands), the membranes were incubated with the secondary antibody for 1 h at room temperature with the secondary antibody (1:25,000 dilution, donkey anti rabbit CW800). Then the membranes were washed again 3 times with PBS-0.1%T and three times with PBS. Reactive proteins were visualized using a near-infrared imager (Oddyssey detection system, Licor Westburg, Leusden, The Netherlands) and the protein levels were determined by densitometric analysis of the specific protein bands (Image J). GAPDH (Millipore, Merck Chemicals, Amsterdam, The Netherlands) was used as an internal control. Actin or GAPDH served as loading control for all Western blot experiments. After normalization to loading control, sGCα1 and b1 protein data were expressed as mean fold change relative to the concentration in healthy brain tissue, which was set to 1.

PCR studies

Total RNA was prepared with a Miccra D-8 power homogenizer (ART) using the TRIzol reagent® (Invitrogen) and was quantified spectrophotometrically. Then, 1 µg of total RNA were reversely transcribed with the TaqMan® Reverse Transcription Reagents (Applied Biosystems) according to the manufacturer’s protocol using random hexamers. Relative gene expression levels of interleukin(Il)-1ß (assay ID: Mm 00434228_m1, Applied Biosystems) and tumor necrosis factor(Tnf)α (assay ID: Mm 00443258_m1, Applied Biosystems) were quantified with the fluorescent TaqMan® technology. Gapdh (TaqMan® Predeveloped Assay Reagents for gene expression, part number: 4352339E, Applied Biosystems) was used as an endogenous control to normalize the amount of sample RNA. The PCR was performed with equal amounts of cDNA in the StepOnePlusTM Real-Time PCR System (Applied Biosystems) using the TaqMan® Universal 2× PCR Master Mix (Applied Biosystems). Reactions (total volume 12.5 µl) were incubated at 50 °C for 2 min, at 95 °C for 10 min followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. Water controls were included to ensure specificity. Each sample was measured in triplicate and data points were examined for integrity by analysis of the amplification plot. The comparative Ct method was used for relative quantification of gene expression.

Rat hippocampal slice experiments

MTT (3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolim bromide) from Sigma (Madrid, Spain), KT5823 from Tocris (Biogen Científica, Madrid, Spain) and BAY60-2770 as well as BAY58-2776 from Maastricht University (The Netherlands). Adult male Sprague-Dawley rats (275–325 g) from a colony of our animal quarters were used. Experimental procedures were approved by the institutional Ethics Committee of Universidad Autónoma de Madrid (Spain) in accordance with the European Guidelines for the use and care of animals for research. All efforts were made in order to reduce animal suffering and decrease the number of animals used. For hippocampal slices preparation and induction of oxygen and glucose deprivation (OGD), animals were decapitated under sodium pentobarbital anesthesia (60 mg/kg, i.p.), the brains were removed and located into ice-cold Krebs bicarbonate dissection buffer (pH 7.4) composed of: NaCl 120 mM, KCl 2 mM, NaHCO 3 26 mM, KH 2 PO 4 1.18 mM, MgSO 4 10 mM, CaCl 0.5 mM, glucose 11 mM and sucrose 200 mM. The solutions were pre-bubbled with 95% O 2 /5% CO 2 for not less than 30 min before starting the experiment. Thereafter, hippocampi were dissected and immersed in cold, oxygenated dissection buffer before sectioning in transverse slices of 250 µM using a McIlwain Tissue Chopper. Attempting to stabilize tissue after slicing trauma, slices were transferred to sucrose-free dissection buffer, bubbled with 95% O 2 /5% CO 2 , during 45 min at 34 °C. Then, control group slices were incubated 15 min in a Krebs solution, containing (in mM): NaCl 120, KCl 2, NaHCO 3 26, CaCl 2, KH 2 PO 4 1.18, MgSO 4 1.19 and glucose 11 mM, which was equilibrated with 95% O 2 /5% CO 2. Slices subjected to oxygen and glucose deprivation were incubated in a Krebs buffer pre-bubbled with 95% N 2 /5% CO 2 , where 2-deoxyglucose replaced glucose. OGD period was followed by 120 min of reoxygenation at 37 °C where slices were returned back to an oxygenated Krebs solution containing glucose. During the reoxygenation period, slices were treated with different concentrations of BAY60-2770 or BAY58-2667. KT-5823 was used as an inhibitor of PKG. Cellular viability was quantified by their ability to reduce MTT.60 After the reoxygenation period, slices were incubated with MTT (0.5 mg/ml) in Krebs bicarbonate solution for 40 min at 37 °C. Cell active dehydrogenases can cleave the tetrazolium ring of MTT in order to generate a precipitated formazan. The formazan produced was solubilized by adding 200 µl dimethyl sulfoxide (DMSO), giving rise to a colored compound whose optical density was measured in an ELISA microplate reader at 540 nm. Absorbance values obtained in control slices were taken as 100% viability.

Statistical analysis

All results obtained from stroke brains were analyzed using the GraphPad Prism 5.0 software (GraphPad Software Inc., San Diego, CA, USA). Data were expressed as the means ± standard error of the mean of separate experiments (n = 4). Statistical comparisons between groups were performed using one-way ANOVA, followed by a two-tailed unpaired Student's t test (for sGC alpha1 subunit). Differences between two treatments were considered significant at P < 0.05. All results were expressed as mean ± SEM except for ordinal functional outcome scales that were depicted as scatter plots. Numbers of animals (N = 10) necessary to detect a standardized effect size on infarct volumes ≥ 0.2 (vehicle treated control mice vs. BAY60-2770 treated mice) were determined via a priori sample size calculation with the following assumptions: α = 0.05, ß = 0.2, mean, 20% SD of the mean (GraphPad Stat Mate 2.0; GraphPad Software). Data were tested for Gaussian distribution with the D’Agostino and Pearson omnibus normality test and then analyzed by one-way analysis of variance (ANOVA) with posthoc Bonferroni adjustment for P values. If only 2 groups were compared, unpaired, two-tailed Student's t test was applied. Nonparametric functional outcome scores were compared by Kruskal–Wallis test with posthoc Dunn multiple comparison test. For comparison of survival curves the log-rank test was used. P values < 0.05 were considered statistically significant.

Data availability

Experimental data from the cGMP-related cluster within the human diseasome are available in the supplemental tables S1,S2A–D and S3 from the authors. Relevant experimental data are available from the authors.