Pentraxin 3 promotes long-term cerebral blood flow recovery, angiogenesis, and neuronal survival after stroke
Restoration of cerebral blood flow (CBF) and upregulation of angiogenesis are crucial for brain repair and functional recovery after cerebral ischaemia. Pentraxin 3 (PTX3) is a key regulator of angiogenesis and is emerging as a promising target for cerebrovascular repair after stroke. Here, we investigated for the first time the role of PTX3 in long-term CBF, angiogenesis, and neuronal viability after ischaemic stroke induced by transient middle cerebral artery occlusion (MCAo). Lack of PTX3 had no effect on early brain damage, but significantly impaired restoration of CBF, 14 and 28 days after MCAo, compared to wild-type (WT) mice. Immunohistochemical analysis revealed that PTX3 KO mice have significantly greater neuronal loss, significantly decreased vessel diameter, vessel proliferation, vascular density, and reactive astrocytes and decreased expression of vascular endothelial growth factor receptor 2 (VEGR2), vascular extracellular matrix (ECM)-proteins (collagen IV, laminin), and integrin-β, in the ipsilateral (stroke) hemisphere compared to WT mice, 28 days after MCAo. Therefore, PTX3 promotes sustained long-term recovery of CBF, angiogenesis, and neuronal viability after cerebral ischaemia. Collectively, these findings demonstrate the potential and clinical relevance of PTX3 as a promising therapeutic target, providing sustained long-term post-stroke neurovascular repair and reducing the loss of neurons.
Pentraxin 3 (PTX3) is a key regulator of angiogenesis and is emerging as a promising target for cerebrovascular repair after stroke.
Restoration of cerebral blood flow (CBF) and angiogenesis are crucial for brain repair and functional recovery after cerebral ischaemia.
PTX3 promotes sustained long-term recovery of CBF, angiogenesis, and neuronal viability after cerebral ischaemia.
KeywordsIschaemic stroke Cerebral blood flow Angiogenesis Vascular remodelling Neuroprotection
A rapid and sustained re-establishment of normal cerebral blood flow (CBF) after ischaemic stroke (known as reperfusion) is associated with a better outcome and improved functional recovery. Indeed, clinical data suggests that ischaemic stroke patients that exhibit greater CBF show neurological improvements , whereas pre-clinical evidence indicates that a reduction in CBF post-stroke is associated with increased brain damage in rats . Furthermore, experimental studies have reported that angiogenesis leads to the formation of new blood vessels after ischaemia and promotes recovery .
A new regulator of angiogenesis is the acute phase protein pentraxin 3 (PTX3), the function of which in cardiovascular and cerebrovascular disease is emerging [4, 5]. We have previously shown that brain PTX3 expression is upregulated after experimental stroke in the ipsilateral (stroke) hemisphere in mice, promoting recovery in the brain by reducing oedema and improving blood-brain barrier (BBB) integrity by enhancing glial scar formation . Furthermore, previous studies have reported a reduction in the number of capillaries in PTX3 knockout (KO) mice in a model of acute myocardial infarction . Published data from our group suggests that PTX3 promotes angiogenesis 14 days after experimental stroke, demonstrated by a decrease in vasculature, fewer newly formed blood vessels, and reduced expression of vascular endothelial growth factor receptor 2 (VEGFR2) in PTX3 KO mice . Despite these findings, the effect of PTX3 on CBF is currently completely unknown, and the mechanisms underlying PTX3 regulation of post-stroke angiogenesis remain incompletely understood. In addition, whether pro-angiogenic actions of PTX3 are a transient or sustained long-term response is currently unknown.
Here, we show for the first time that PTX3 promotes sustained long-term recovery of CBF after experimental cerebral ischaemia. Crucially, we demonstrate a long-lasting pro-angiogenic effect of PTX3, revealed by a reduction in vessel diameter, proliferation of vessels, cerebrovasculature, and VEGFR2 expression, in PTX3 KO mice compared to wild-type (WT) mice, 28 days after stroke. PTX3 also upregulates vascular integrin-β1 expression in the stroke region of WT mice, but not in PTX3 KO mice. Moreover, integrin-β1 expression is significantly reduced compared to WT mice in the ipsilateral hemisphere. Interestingly, our study indicates that PTX3 promotes expression of both vascular and peri-vascular reactive (glial fibrillary acidic protein (GFAP) positive (+)) astrocytes in ischaemic regions, which was previously unknown. Finally, we show that PTX3 promotes long-term neuronal viability after experimental stroke. Collectively, our results suggest that PTX3 may be an effective post-stroke treatment option reducing neuronal loss and promoting long-term repair.
PTX3 KO mice and WT littermates were from heterozygote mice (obtained from Dr. Cecilia Garlanda, Humanitas Clinical and Research Center, Rozzano, Italy) and were genotyped as described previously . All mice were bred on a C57BL/6 background. All animal procedures were carried out in accordance with the Animal Scientific Procedures Act (1986) and the European Council Directive 2010/63/EU and were approved by the Home Office and Animal Welfare and Ethics Review Board, University of Manchester (UK). All experiments adhered to the ARRIVE guidelines and IMPROVE guidelines [8, 9]. Surgical procedures and behavioural tests were carried out by an experimenter blinded to the genotype.
Cerebral ischaemia induced by transient middle cerebral artery occlusion (MCAo)
Induction of anaesthesia was achieved by inhalation of 4% isoflurane (30% oxygen and 70% nitrous oxide gas, AbbVie Ltd., UK) and was maintained at 1.75% isoflurane. During surgery, a rectal probe was used to assess body temperature, which was kept at 37 °C ± 0.5 °C with a heat blanket (Harvard Apparatus, Edenbridge, Kent, UK). A laser Doppler monitor (Oxford Optronix, Abingdon, UK) was used to confirm a drop in CBF in the middle cerebral artery (MCA) region following occlusion and subsequent recovery of CBF. Focal cerebral ischaemia was achieved by MCAo using a protocol described previously as follows; prior to incision, topical anaesthetic (EMLA, 5% prilocaine and lidocaine, AstraZeneca, UK) was administered on to the areas of skin requiring incision. A laser-Doppler probe located 6-mm lateral and 2-mm posterior from bregma was fixed on to the skull with vetbond. An incision was made into the middle front section of the neck, and the right common carotid artery was exposed and ligated. The internal carotid artery received temporary ligation. An incision in to the common carotid artery was made, followed by the insertion and advancement of a 6–0 monofilament (Doccol, Sharon, MA, USA) through the internal carotid artery. After 15 min of occlusion (a short occlusion time was used to reduce mortality), the filament was removed allowing reperfusion. The neck wound was sutured, and saline (0.5 ml, for rehydration) and buprenorphine (0.05 mg/kg, providing general analgesia) were administered subcutaneously (sc). Animals were allowed to recover in an incubator (26 °C) and then held in ventilated cages placed on a heat pad (24 °C) under standard laboratory conditions with ad libitum access to mashed food and water for 24 h. Finally, cages were returned to their home rack and kept under normal housing conditions. All animals in our studies demonstrated a minimum of 70% decrease in CBF due to ischaemia. For the 28-day study, three WT and four PTX3 KO mice were culled at 24 h post-surgery due to ischaemic damage deemed too severe as per the IMPROVE guidelines. For the 48 h time-point study, one WT mouse was culled at 24 h due to severe ischaemic damage. Furthermore, one WT and one PTX3 KO mice from the laser speckle study were culled at 24 h post-surgery due to ill health .
Laser speckle contrast imaging (LSCI) and analysis
Mice were anaesthetized with inhalation of 4% isoflurane and placed in a stereotaxic frame (World Precision Instruments, USA) on a heat mat set to 37 °C, positioned under a moorFLPI2 Full-Field Perfusion Imager (Moor instruments, UK). Anaesthesia was maintained at 1.75% isoflurane. The skin on top of the skull was opened to expose the skull bone, which was kept intact, and LSCI was conducted for 2 min at 100 frames (4 s per frame). Mice were imaged prior to MCAo (baseline recording), 72 h, 14 days, and 28 days post-MCAo. Two regions of interest (ROIs) located in the primary MCA area and frontal cortical regions were identified and used for analysis. To evaluate CBF, 15 images per mouse for each time point were analysed using moorFLPI2 Full-Field Laser Perfusion Imager Review V5.0 software. For all animals, flux values at each time point were measured for each ROI in the ipsilateral and corresponding ROIs in the contralateral hemisphere, and ipsilateral flux was expressed as a % of contralateral flux. Analysis was conducted under blinded conditions.
Animals were anaesthetised with 4% isoflurane and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde (PFA) 48 h, 7 days, 14 days, or 28 days after MCAo surgery (see Supplemental materials for more detail).
Infarct volume analysis
Immunohistochemistry was carried out on free floating brains (see Supplemental materials for more detail).
Microscopy and image analysis
High power field images were collected on an Olympus BX51 upright microscope using a ×20 objective and captured using Coolsnap ES camera (photometrics, USA) through MetaVue software (Molecular Devices, USA). Images were then analysed with ImageJ software (National Institutes of Health, USA). For each animal, immunohistochemistry micrographs were analysed on coronal sections of the same co-ordinates (approximately bregma level 0.84 mm, according to mouse brain atlas, www.mouse.brain-map.org), which represented the middle of the infarcted territory from anterior-posterior perspective. Both ipsilateral and contralateral hemispheres in penumbra and core regions were measured (see Supplemental Fig. 1 for diagram). The % area of the total image stained with PECAM-1, VEGFR2, lectin, col IV, laminin, integrin-β1, and GFAP were quantified with ImageJ. In addition, VEGFR2 was normalised to PECAM-1, and col IV, laminin, and integrin-β1 were normalised to lectin (e.g. (% area staining col IV / % area staining lectin) × 100). Vessel diameter (μM) of col IV labelled vessels, the number of vascular GFAP+ astrocytes, peri-vascular GFAP+ astrocytes, vessels, KI-67+ vessels, and NeuN+ neurons were counted manually with ImageJ. For vessel diameter analysis, the vessel diameter of all vessels present in an image were analysed, and an overall mean vessel diameter was then calculated. The width of the centre of the vessel in μM was measured using ImageJ software. In instances where vessels were composed of multiple branches, the width of the centre of each branch was measured and an average value was calculated. Four images, i.e. 1 per region, per mouse were analysed. Number of KI-67+ vessels was also expressed as a % of total number of vessels. Iba1+ microglia were counted based on their activation status determined by their morphology as described previously . All collection and analyses of images were carried out by a blinded experimenter.
All data are presented as mean values ± standard deviation of the mean (SD). For LSCI analyses, linear mixed modelling was used to evaluate the effect of independent factors (genotype and time) on the dependent variable . All factors and interactions were modelled as fixed effects. A within-subject design with random intercepts was used for all models. The significance of inclusion of a dependent variable or interaction terms was evaluated using log-likelihood ratio. Holm-Sidak post hocs were then performed for planned pair-wise comparisons using the least square means . Homoscedasticity and normality were evaluated graphically using predicted vs residual and Q-Q plots, respectively. All analyses were performed using R (version 3.3.3). For the remaining data, normality was assessed with the Shapiro-Wilk test, and appropriate transformations were employed when necessary. Statistical analyses performed on normally distributed data were unpaired Student’s t test and repeated measures two-way analysis of variance (ANOVA) test with Sidak corrected post hoc test. The Mann-Whitney test was used for data that were not normally distributed. Accepted levels of significance were *P < 0.05, **P < 0.01, and ***P < 0.001. Statistical analyses were carried out using GraphPad Prism 7.0.
PTX3 KO mice exhibit impaired long-term recovery of cerebral blood flow after ischaemic stroke
Lack of PTX3 impairs vascular remodelling after cerebral ischaemia
PTX3 is important for driving vessel-associated cellular proliferation 14 and 28 days post-stroke
PTX3 KO mice display impaired angiogenesis 28 days after experimental stroke
PTX3 upregulates integrin-β1 expression in the cerebrovasculature 28 days after MCAo
Post-stroke expression of reactive GFAP+ astrocytes is promoted by PTX3
The number of both peri-vascular (1.74-fold) and vascular (1.40-fold) GFAP+ astrocytes was found to be significantly greater in the ipsilateral hemisphere compared to the contralateral hemisphere in WT mice, but not in PTX3 KO mice (Fig. 6b). Moreover, PTX3 KO mice exhibited a significantly smaller number of both peri-vascular (50%) and vascular (53%) GFAP+ astrocytes in their ipsilateral hemisphere, when compared to WT ipsilateral hemisphere. In contrast, although both WT and PTX3 KO mice demonstrated significantly increased number % and mean activation score of activated microglia, in their ipsilateral hemisphere compared to contralateral hemisphere, our data indicated no significant difference between genotypes (Fig. 6c). Consequently, these results demonstrate for the first time another potential post-stroke pro-angiogenic mechanism driven by PTX3 involving reactive astrocytes.
PTX3 reduces neuronal loss 28 days after experimental stroke
To our knowledge, the role of the acute phase protein PTX3 on CBF after cerebral ischaemia has not been previously studied, and here, we are the first to show that PTX3 is a key mediator of long-term CBF recovery. Specifically, we demonstrated that in the primary MCA area and frontal cortical region, PTX3 KO mice showed no recovery in CBF. In contrast, mice expressing PTX3 showed a marked increase in CBF at 14 days and significantly increased CBF 28 days after stroke, when compared to CBF levels at 72 h. Importantly, in WT mice, CBF was close to baseline levels at 14 days and returned to baseline values by 28 days. In addition, vascular remodelling (i.e. increased vessel diameter) occurs in the ischaemic hemisphere of WT mice, but not PTX3 KO mice, after cerebral ischaemia. These observations are consistent with a previous report showing that vessel diameter increases alongside recovery of CBF during the sub-acute phase after cerebral ischaemia . Therefore, we believe that the increased vessel diameter and/or neoangiogenesis observed in WT mice in our study may have contributed to the increased CBF we observed in these mice. It is possible that these neosynthesised vessels are perfused; however, this requires further experimental investigation. Collectively, our findings are clinically important as they strongly suggest that PTX3 aids long-term recovery of CBF after cerebral ischaemia, possibly mediated by mechanisms of ECM remodelling and angiogenesis, and therefore that upregulating PTX3 is a promising therapeutic strategy to improve outcome in stroke patients.
There is robust evidence demonstrating that endothelial cell proliferation, which is a key angiogenic response, directly leads to an increase in vessel diameter [27, 28]. Our study further demonstrated that lack of PTX3 significantly impaired long-term angiogenesis after cerebral ischaemia. Indeed, vessel-associated cell proliferation was markedly decreased in PTX3 KO mice compared to WT mice, and vessel density and vascular VEGFR2 expression were also significantly decreased in PTX3 KO mice compared to WT mice, up to 28 days after MCAo. These data build on our previous finding that PTX3 KO mice have impaired angiogenesis 14 days after cerebral ischaemia , but show for the first time that long-term (28 days) post-stroke angiogenesis regulated by PTX3 correlates with total recovery of CBF. A previous study has reported that an acidic microenvironment, similar to acidosis pH levels observed in ischaemic stroke, activates PTX3 into a tissue remodelling and repair mode in a lung injury model . The remodelling of the ECM has been described previously as an important factor regulating vessel diameter and angiogenesis . In particular, integrin-β1 is critically important for vessel formation and integrity, and studies blocking integrin-β1 have demonstrated a significant reduction in vessel diameter [17, 30]. Our study also found that expression of vessel-associated integrin-β1, as well as ECM proteins col IV and laminin, and perivascular astrocytes were all reduced by the lack of PTX3, 28 days after cerebral ischaemia, further suggesting that cerebrovascular ECM remodelling regulated by PTX3 may be a key step for restoration of CBF after stroke. Interestingly, our study also found that PTX3 KO mice exhibit a significant decrease in neuronal integrity compared to WT mice 28 days after MCAo. A previous study already demonstrated the acute neuroprotective effect of PTX3 in a mouse model of seizure . Lack of PTX3 had no effect on acute neurodegeneration in our study and as previously reported by us , but our data suggest that PTX3 may support neuronal survival during the sub-acute phase of stroke. Microglia are key contributors to neuronal survival in the injured brain and a recent report demonstrated protective action of microglia early after stroke . We found that activated microglia are present in ischaemic hemispheres of both WT and PTX3 KO mice, although no significant difference was observed between genotypes, suggesting that PTX3 may support long-term neuronal survival independently of microglial activity. We did not observe any functional behavioural differences between WT and PTX3 KO mice. The discrepancy between structural and functional outcome in our study may suggest that better refined behavioural tests are needed in order to investigate the role of PTX3 on functional recovery.
In summary, we have shown that PTX3 is a key promoter of sustained long-term CBF recovery, angiogenesis, and neuronal viability 28 days after cerebral ischaemia, demonstrating its beneficial clinical potential. We propose that PTX3 can be targeted to promote long-term neurovascular repair and protect neurons after ischaemic stroke and possibly other cerebrovascular ischaemic injuries.
The authors would like to thank the Bioimaging Facility in the Faculty of Biology, Medicine, and Health at the University of Manchester for equipment and advice in imaging.
IR performed the genotyping of animals, LSCI, tissue processing, histology, data analysis, and wrote the manuscript. RW conducted the surgeries and contributed to histology and writing of the manuscript. EL contributed to LSCI and advised analysis. JRA provided essential statistical analysis and contributed to the writing of the manuscript. OR provided support in tissue processing and animal welfare. CG provided the mice. EP and SA devised the study, obtained funding, and contributed to writing of the manuscript.
This work was supported by funding from the British Heart Foundation (Reference: FS/14/70/31006) and the Medical Research Council (Reference: MR/N003586/10).
Compliance with ethical standards
Conflict of interests
The authors declare that there is no conflict of interest.
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