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Traumatic brain injury leads to cellular damage which in turn results in the rapid release of damage-associated molecular patterns (DAMPs) that prompt resident cells to release cytokines and chemokines. These in turn rapidly recruit neutrophils, which assist in limiting the spread of injury and removing cellular debris. Microglia continuously survey the CNS (central nervous system) compartment and identify structural abnormalities in neurons contributing to the response. After some days, when neutrophil numbers start to decline, activated microglia and astrocytes assemble at the injury site—segregating injured tissue from healthy tissue and facilitating restorative processes. Monocytes infiltrate the injury site to produce chemokines that recruit astrocytes which successively extend their processes towards monocytes during the recovery phase. In this fashion, monocytes infiltration serves to help repair the injured brain. Neurons and astrocytes also moderate brain inflammation via downregulation of cytotoxic inflammation. Depending on the severity of the brain injury, T and B cells can also be recruited to the brain pathology sites at later time points.

Cerebral microdialysis (CMD) is used in severe traumatic brain injury (TBI) in order to recover metabolites in brain extracellular fluid (ECF). To recover larger proteins and avoid fluid loss, albu...

Following traumatic brain injury (TBI), raised cerebral lactate/pyruvate ratio (LPR) reflects impaired energy metabolism. Raised LPR correlates with poor outcome and mortality following TBI. We prospectively recruited patients with TBI requiring neurocritical care and multimodal monitoring, and utilised a tiered management protocol targeting LPR. We identified patients with persistent raised LPR despite adequate cerebral glucose and oxygen provision, which we clinically classified as cerebral ‘mitochondrial dysfunction’ (MD). In patients with TBI and MD, we administered disodium 2,3-13C2 succinate (12 mmol/L) by retrodialysis into the monitored region of the brain. We recovered 13C-labelled metabolites by microdialysis and utilised nuclear magnetic resonance spectroscopy (NMR) for identification and quantification. Of 33 patients with complete monitoring, 73% had MD at some point during monitoring. In 5 patients with multimodality-defined MD, succinate administration resulted in reduced LPR(−12%) and raised brain glucose(+17%). NMR of microdialysates demonstrated that the exogenous 13C-labelled succinate was metabolised intracellularly via the tricarboxylic acid cycle. By targeting LPR using a tiered clinical algorithm incorporating intracranial pressure, brain tissue oxygenation and microdialysis parameters, we identified MD in TBI patients requiring neurointensive care. In these, focal succinate administration improved energy metabolism, evidenced by reduction in LPR. Succinate merits further investigation for TBI therapy.

Background Flow aneurysms (FAs) associated with brain arteriovenous malformations (AVMs) are thought to arise from increased hemodynamic stress due to high-flow shunting. This study aims to describe the changes in conservatively managed FAs after successful AVM treatment. Methods Patients with symptomatic AVMs and associated FAs who underwent successful treatment of the AVM between 2008 and 2017 were included. FA dimensions were measured on surveillance angiography to assess longitudinal changes. Results Thirty-two patients were identified with 48 FAs. Sixteen (33%) FAs were treated endovascularly; 18 (38%) FAs were treated surgically; and 14 (29%) FAs (11 patients) were monitored. FAs demonstrated a decrease in size from 5.0 mm to 3.8 mm (24%; P = 0.016) and 4.9 mm to 3.6 mm (27%; P = 0.013) in height and width, respectively, over a median 35 months. However, on subgroup analysis, only class IIb aneurysms demonstrated a significant decrease in size (51% reduction in largest diameter, P = 0.046) and only 3 FAs (21%) resolved. There were no hemorrhages observed during follow-up. Conclusions While conservatively managed FAs demonstrated a reduction in size after the culprit AVM was treated, this was only significant in FAs located close to an AVM nidus (class IIb). There were no hemorrhages during the median 35 months’ follow-up; however, long-term data are lacking. Our data support close observation of all conservatively managed aneurysms and a tailored approach based on the proximity to the nidus and observed changes in size.

Background Specific procedural complications in aneurysm surgery are broadly related to vascular territory compromise and brain/nerve retraction; vascular complications account for about half of this. Intraoperative indocyanine green video angiography (ICG-VA) provides real-time high spatial resolution imaging of the cerebrovascular architecture, allowing immediate quality assurance of aneurysm occlusion and vessel integrity. The aim of this study was to examine whether the routine use of ICG-VA reduced early procedural complications related to vascular compromise or injury during micro-neurosurgical clipping of ruptured cerebral aneurysms. Methods Retrospective comparative observational study of 412 adult good-grade (WFNS 1 or 2) SAH patients who had undergone microsurgical clipping without (n = 200, 2001–2004) or with (n = 212, 2009–2015) ICG-VA in a high-volume neurosurgical centre. Results The ICG-VA group had a significantly lower incidence of procedural vascular complications (7/212; 3.3%) compared with the non-ICG-VA group (19/200; 9.5%) (Fisher’s exact test p = 0.0137). Conclusions ICG-VA is a straightforward, easy-to-use, intraoperative adjunct which significantly reduces avoidable ‘technical error’ related morbidity.

Background Cranioplasty is an increasingly common procedure performed in neurosurgical centres following a decompressive craniectomy (DC), however, timing of the procedure varies greatly. Objectives The aim of this study is to compare the surgical outcomes of an early compared to a late cranioplasty procedure. Methods Ninety adult patients who underwent a prosthetic cranioplasty between 2014 and 2017 were studied retrospectively. Timing of operation, perioperative complications and length of stay were assessed. Early and late cranioplasties were defined as less or more than 3 months since craniectomy respectively. Results Of the 90 patients, 73% received a late cranioplasty and 27% received an early cranioplasty. The median interval between craniectomy and cranioplasty was 13 months [range 3–84] in late group versus 54 days [range 33–90] in early group. Twenty-two patients in the early group (91%) received a cranioplasty during the original admission while undergoing rehabilitation. Complications were seen in 25 patients (28%). These included wound or cranioplasty infection, hydrocephalus, symptomatic pneumocephalus, post-operative haematoma and cosmetic issues. The complication rate was 21% in the early group and 30% in the late group (P value 0.46). There was no significant difference in the rate of infection or hydrocephalus between the two groups. Length of stay was not significantly increased in patients who received an early cranioplasty during their initial admission (median length of stay 77 days versus 63 days, P value 0.28). Conclusion We have demonstrated the potential for early cranioplasty to be a safe and viable option, when compared to delayed cranioplasty.

Neuroinflammation has been shown to mediate the pathophysiological response following traumatic brain injury (TBI). Accumulating evidence implicates astrocytes as key immune cells within the central nervous system (CNS), displaying both pro- and anti-inflammatory properties. The aim of this study was to investigate how in vitro human astrocyte cultures respond to cytokines across a concentration range that approximates the aftermath of human TBI. To this end, enriched cultures of human induced pluripotent stem cell (iPSC)-derived astrocytes were exposed to interleukin-1β (IL-1β) (1–10,000 pg/mL), IL-4 (1–10,000 pg/mL), IL-6 (100–1,000,000 pg/mL), IL-10 (1–10,000 pg/mL) and tumor necrosis factor (TNF)-α (1–10,000 pg/mL). After 1, 24, 48 and 72 h, cultures were fixed and immunolabeled, and the secretome/supernatant was analyzed at 24, 48, and 72 h using a human cytokine/chemokine 39-plex Luminex assay. Data were compared to previous in vitro studies of neuronal cultures and clinical TBI studies. The secretome revealed concentration-, time- and/or both concentration- and time-dependent production of downstream cytokines (29, 21, and 17 cytokines, respectively, p

Introduction The response throughout the National Health Service (NHS) to the first wave of coronavirus disease 2019 (COVID-19) was substantial. With repeat winter waves of COVID-19 or other viral pathogens in the future being likely, we sought to review the impact of the response to the first wave on the delivery and training of neurosurgery in a large tertiary neurosurgical centre. Methods We performed a retrospective review over the three-month period of 8th March to 7th June 2020 as indicative of the peak of the pandemic in the UK. For referrals, our online referral portal was reviewed (ORION). For admissions and operations, electronic patient records were reviewed (EPIC systems). Trust wide update emails and policies were also reviewed. Results In response to the pandemic, neurosurgical service provision was severely restricted in the early days of the pandemic in the form of reduced beds, medical and nursing staff and theatres. This was rapidly realised to be unsustainable, and resources were slowly reopened although not to pre-COVID-19 levels. Although referrals did not substantially reduce, the number of elective and emergency admissions, length of stay, theatre efficiency and operative numbers (by pathology and grade of operating surgeon) did significantly reduce except for emergencies performed by consultants. If similar trainee operating numbers would have persisted, this would lead to a significant delay to completion of training. Conclusions All aspects of neurosurgical provision were detrimentally affected due to the rapid response to the first wave of COVID-19 in our institution. Such repeated reductions in acute services such as neurosurgery would be unsustainable. It is pertinent to re-visit these effects in preparation for future infection waves to better protect acute neurosurgical services.

Navigation is emerging as a useful adjunct in percutaneous, minimally invasive spinal surgery (MIS). The aim of this study was to compare C-Arm navigated, O-Arm navigated and conventional 2D-fluoroscopy assisted MIS thoracic and lumbosacral spine fixation techniques in terms of operating time, radiation exposure and accuracy of pedicle screw (PS) placement. Retrospective observational study of 152 consecutive adults who underwent MIS fixations for spinal instability: 96 2D-fluoroscopy assisted, 39 3D-C-Arm navigated and 27 using O-Arm navigated. O-Arm navigation significantly reduced PS misplacement (1.23%, p) compared to 3D-C-Arm navigation (7.29%, p = 0.0082) and 2D-fluoro guided placement (5.16%, p = 0379). 3D-C-Arm navigation was associated with lower procedural radiation exposure of the patient (0.4 mSv) than O-Arm navigation (3.24 mSv) or 2D-fluoro guidance (1.5 mSv). Operative time was comparable between three modalities. O-Arm navigation provides greater accuracy of percutaneous instrumentation placement with an acceptable procedural radiation dose delivered to the patients and comparable operative times. These slides can be retrieved under Electronic Supplementary material.

Case report. To investigate the feasibility of using two independent image guidance systems to simultaneously fix multiple segment spine fractures. Image guidance is increasingly used to aid spinal fixation. We describe the first use of multiple navigation systems during a single procedure allowing for multi-segment spinal fixations to be performed simultaneously and capitalizing the advantages of navigation. Two Medtronic Stealth Station S7™ systems with O-arm image capture were used to guide fixation of C6 and T12, unstable, AO A4, three-column fractures, in a patient with ankylosing spondylitis. Two surgical teams were able to perform cervico-thoracic and thoraco-lumbar fixations simultaneously. Operative time was 2.5 h. Post-operative imaging showed accurate instrumentation placement. The patient recovered without any neurological sequelae. Optical independence of the Medtronic Stealth Station™ system allowed for simultaneous navigation guided fixation of multiple segment fractures without compromising accuracy. This may result in shortened operative time and morbidity associated with prolonged prone positioning of polytrauma patients, as well as reducing radiation exposure for theatre staff.

Spinal cord injury (SCI) is a life-changing event not only for the patient but also for the whole family. Both patient and families need physical and psychological support from the time of injury and throughout their life with SCI. Once the acute stage is over this support is provided by the inpatient rehabilitation team during their inpatient stay and then by the community teams as well as various voluntary organizations. Rehabilitation in a specialized SCI centre is key to achieving maximum independence following the injury. Successful discharge into the community is complex and involves coordinated efforts from the multidisciplinary team, social care team, families, voluntary organizations, as well as legal teams. SCI rehabilitation in this chapter is divided into two phases; acute and chronic.

Background: Two randomised trials assessing the effectiveness of decompressive craniectomy (DC) following traumatic brain injury (TBI) were published in recent years: DECRA in 2011 and RESCUEicp in 2016. As the results have generated debate amongst clinicians and researchers working in the field of TBI worldwide, it was felt necessary to provide general guidance on the use of DC following TBI and identify areas of ongoing uncertainty via a consensus-based approach. Methods: The International Consensus Meeting on the Role of Decompressive Craniectomy in the Management of Traumatic Brain Injury took place in Cambridge, UK, on the 28th and 29th September 2017. The meeting was jointly organised by the World Federation of Neurosurgical Societies (WFNS), AO/Global Neuro and the NIHR Global Health Research Group on Neurotrauma. Discussions and voting were organised around six pre-specified themes: (1) primary DC for mass lesions, (2) secondary DC for intracranial hypertension, (3) peri-operative care, (4) surgical technique, (5) cranial reconstruction and (6) DC in low- and middle-income countries. Results: The invited participants discussed existing published evidence and proposed consensus statements. Statements required an agreement threshold of more than 70% by blinded voting for approval. Conclusions: In this manuscript, we present the final consensus-based recommendations. We have also identified areas of uncertainty, where further research is required, including the role of primary DC, the role of hinge craniotomy and the optimal timing and material for skull reconstruction. publishedVersion

OBJECTIVEIntracranial hypertension and impaired cerebral autoregulation are common causes of secondary injuries in patients with traumatic brain injury (TBI). The primary outcome of this study was to assess whether a noninvasive method to estimate intracranial pressure (ICP) based on the ultrasonography of the optic nerve sheath diameter (ONSD) measured at the time of neurocritical care unit (NCCU) admission is correlated with the mean ICP during NCCU stay. Secondary outcomes were to assess whether ONSD is correlated with the dose of ICP > 20 mm Hg and impaired autoregulation during NCCU stay and with instantaneous ICP and whether ONSD is associated with NCCU mortality.METHODSThis prospective observational monocentric study included adults with severe TBI. ONSD was measured at NCCU admission, immediately after invasive ICP insertion. ONSD-predicted noninvasive ICP (nICPONSD) was calculated according the formula: nICPONSD = 5 × ONSD − 14 (nICPONSD in mm Hg, ONSD in mm). Autoregulation was measured using the pressure reactivity index (PRx).RESULTSIn total, 100 patients were included in this study. ONSD was significantly correlated with mean ICP (r = 0.46, p < 0.0001), with mean PRx (r = 0.21, p = 0.04), and with the dose of ICP > 20 mm Hg during NCCU stay (r = 0.49, p < 0.0001). Admission nICPONSD was shown to be significantly correlated with instantaneous ICP (r = 0.85, p < 0.001). ONSD at admission was significantly correlated with NCCU mortality (p = 0.02).CONCLUSIONSONSD measured at NCCU admission can give important information about patients at risk of developing intracranial hypertension and impaired autoregulation. ONSD examination could be useful to screen patients at admission to determine who would benefit from further invasive ICP monitoring.

Non-invasive measurement of intracranial pressure (ICP) can be invaluable in the management of critically ill patients. Invasive measurement of ICP remains the "gold standard" and should be performed when clinical indications are met, but it is invasive and brings some risks. In this project, we aim to validate the non-invasive ICP (nICP) assessment models based on arterious and venous transcranial Doppler ultrasonography (TCD) and optic nerve sheath diameter (ONSD).We included brain injured patients requiring invasive ICP monitoring (intraparenchymal or intraventricular). We assessed the concordance between ICP measured non-invasively with arterious [flow velocity diastolic formula (ICPLinear regression showed a positive relationship between nICP and ICP for all the methods, except PIv. ICPOur results demonstrate that among the non-invasive methods studied, ONSD showed the best accuracy in the detection of ICP.

Background: Non-invasive measurement of intracranial pressure (ICP) can be invaluable in the management of critically ill patients. Invasive measurement of ICP remains the “gold standard” and should be performed when clinical indications are met, but it is invasive and brings some risks. In this project, we aim to validate the non-invasive ICP (nICP) assessment models based on arterious and venous transcranial Doppler ultrasonography (TCD) and optic nerve sheath diameter (ONSD).

ObjectivesThe objective of this study was to investigate how in vitro astrocyte cultures respond to cytokine pro- and anti-inflammatory cytokine concentrations, corresponding to those seen in the aftermath of human TBI, by analysing downstream cytokine generation.DesignIn vitro study.SubjectsHuman induced pluripotent stem cells (iPSC)-derived astrocytes.MethodsThe astrocytes were exposed to levels of TNF (1–10,000 pg/ml), IL-6 (100–1,000,000 pg/ml), Interleukin-1β (IL-1β, 1–10,000 pg/ml), Interleukin-4 (IL-4, 1–10,000 pg/ml) and Interleukin-10 (IL-10, 1–10,000 pg/ml). Following 24, 48 and 72 hours, culture supernatant was extracted and analysed using a human cytokine/chemokine 39-plex luminex assay (ThermoFisher).ResultsThe astrocyte secretome revealed concentration-, time- or concentration*time-dependent production of downstream cytokines (12, 8 and 2 cytokines, respectively pConclusionsiPSC-derived astrocytes exposed to cytokine concentrations reflecting those in TBI generate an increased downstream cytokine production, especially when exposed to IL-1β and TNF. This is in contrast to our previous work on neuronal cultures where IL-1β only produced a few down-stream cytokines.1 More work is needed to better understand how different cells in the CNS respond to the neuroinflammatory milieu after TBI alone and in combination.

Previous studies of pulmonary arterial hypertension (PAH) have implicated excessive transforming growth factor (TGF)-β1 signaling and reduced bone morphogenetic protein (BMP) signaling in the disease pathogenesis. Reduced BMP signaling in pulmonary artery smooth muscle cells (PASMCs) from patients with heritable PAH is a consequence of germline mutations in the BMP type II receptor (BMPR-II). We sought to establish whether the TGF-β1 and BMP4 pathways interact in PASMCs, and if this is altered in cells with BMPR-II mutations. Control PASMCs or from patients with PAH harboring BMPR-II mutations were treated with BMP4, TGF-β1, or cotreated with both ligands. Signaling was assessed by examination of Smad phosphorylation, luciferase reporters, and the transcription of BMP4 or TGF-β1-responsive genes. TGF-β1 attenuated BMP4-mediated inhibitors of differentiation 1/2 induction and abolished the response in BMPR-II mutant PASMCs, whereas BMP4 did not alter TGF-β1-mediated transcription. Activin-like kinase 5 inhibition blocked this effect, whereas cycloheximide or pharmacological inhibitors of TGF-β-activated kinase 1, extracellular signal-regulated kinase 1/2, or p38 mitogen-activated protein kinase were ineffective. BMP4 and TGF-β1 cotreatment did not alter the activation or nuclear translocation of their respective Smad signaling proteins. Small interfering RNA for Smad3, but not Smad2, Smad6, or Smad7, reversed the inhibition by TGF-β1. In addition, TGF-β-activated kinase 1 inhibition blocked Smad3 phosphorylation, implying that C-terminal Smad3 phosphorylation is not required for the inhibition of BMP4 signaling by TGF-β1. TGF-β1 reduces BMP4 signaling in PASMCs, a response that is exacerbated on the background of reduced BMP responsiveness due to BMPR-II mutations. These data provide a rationale for therapeutic inhibition of TGF-β1 signaling in PAH.

Isolated oculomotor nerve palsy following head injury is uncommon. It can only be diagnosed with confidence if it is known to have developed immediately following trauma and if adequate investigations exclude secondary causes. The recovery is only partial and this has repercussion on patients' quality of life.

Pulmonary hypertension is a multifactorial disease characterized by sustained elevation of pulmonary vascular resistance (PVR) and pulmonary arterial pressure (PAP). Central to the pathobiology of this disease is the process of vascular remodelling. This process involves structural and functional changes to the normal architecture of the walls of pulmonary arteries (PAs) that lead to increased muscularization of the muscular PAs, muscularization of the peripheral, previously nonmuscular, arteries of the respiratory acinus, formation of neointima, and formation of plexiform lesions. Underlying or contributing to the development of these lesions is hypertrophy, proliferation, migration, and resistance to apoptosis of medial cells and this article is concerned with the cellular and molecular mechanisms of these processes. In the first part of the article we focus on the concept of smooth muscle cell phenotype and the difficulties surrounding the identification and characterization of the cell/cells involved in the remodelling of the vessel media and we review the general mechanisms of cell hypertrophy, proliferation, migration and apoptosis. Then, in the larger part of the article, we review the factors identified thus far to be involved in PH intiation and/or progression and review and discuss their effects on pulmonary artery smooth muscle cells (PASMCs) the predominant cells in the tunica media of PAs.

Background— Hutchinson-Gilford progeria syndrome is a rare inherited disorder of premature aging caused by mutations in LMNA or Zmpste24 that disrupt nuclear lamin A processing, leading to the accumulation of prelamin A. Patients develop severe premature arteriosclerosis characterized by vascular smooth muscle cell (VSMC) calcification and attrition. Methods and Results— To determine whether defective lamin A processing is associated with vascular aging in the normal population, we examined the profile of lamin A expression in normal and aged VSMCs. In vitro, aged VSMCs rapidly accumulated prelamin A coincidently with nuclear morphology defects, and these defects were reversible by treatment with farnesylation inhibitors and statins. In human arteries, prelamin A accumulation was not observed in young healthy vessels but was prevalent in medial VSMCs from aged individuals and in atherosclerotic lesions, where it often colocalized with senescent and degenerate VSMCs. Prelamin A accumulation correlated with downregulation of the lamin A processing enzyme Zmpste24/FACE1, and FACE1 mRNA and protein levels were reduced in response to oxidative stress. Small interfering RNA knockdown of FACE1 reiterated the prelamin A–induced nuclear morphology defects characteristic of aged VSMCs, and overexpression of prelamin A accelerated VSMC senescence. We show that prelamin A acts to disrupt mitosis and induce DNA damage in VSMCs, leading to mitotic failure, genomic instability, and premature senescence. Conclusions— This study shows that prelamin A is a novel biomarker of VSMC aging and disease that acts to accelerate senescence. It therefore represents a novel target to ameliorate the effects of age-induced vascular dysfunction.

Pulmonary hypertension is an orphan disease that until recently has received limited attention within the wider medical community. This has changed distinctly in the last 10 years with the advent of new classes of therapy and a renewed interest in mechanisms of pathogenesis. This review utilized information gathered from recent conferences, and a review of the literature was conducted using MedLine and Pubmed. Accepted mechanisms of pathogenesis and currently available treatments are presented. We will discuss interesting new concepts in pathogenesis, including the importance of genetic forms of the disease and in particular the transforming growth factor receptor superfamily and the evolving evidence of the contribution of dysregulated immunity. Areas of research may yield therapeutic benefits in the not-too-distant future, including anti-proliferative therapies and stem cell therapy.

Mutations in bone morphogenetic protein receptor type II (BMPR-II) underlie most cases of heritable pulmonary arterial hypertension (PAH). However, disease penetrance is only 20-30%, suggesting a requirement for additional triggers. Inflammation is emerging as a key disease-related factor in PAH, but to date there is no clear mechanism linking BMPR-II deficiency and inflammation.To establish a direct link between BMPR-II deficiency, a consequentially heightened inflammatory response, and development of PAH.We used pulmonary artery smooth muscle cells from Bmpr2(+/-) mice and patients with BMPR2 mutations and compared them with wild-type controls. For the in vivo model, we used mice heterozygous for a null allele in Bmpr2 (Bmpr2(+/-)) and wild-type littermates.Acute exposure to LPS increased lung and circulating IL-6 and KC (IL-8 analog) levels in Bmpr2(+/-) mice to a greater extent than in wild-type controls. Similarly, pulmonary artery smooth muscle cells from Bmpr2(+/-) mice and patients with BMPR2 mutations produced higher levels of IL-6 and KC/IL-8 after lipopolysaccharide stimulation compared with controls. BMPR-II deficiency in mouse and human pulmonary artery smooth muscle cells was associated with increased phospho-STAT3 and loss of extracellular superoxide dismutase. Chronic lipopolysaccharide administration caused pulmonary hypertension in Bmpr2(+/-) mice but not in wild-type littermates. Coadministration of tempol, a superoxide dismutase mimetic, ameliorated the exaggerated inflammatory response and prevented development of PAH.This study demonstrates that BMPR-II deficiency promotes an exaggerated inflammatory response in vitro and in vivo, which can instigate development of pulmonary hypertension.

Pulmonary arterial hypertension is the end result of a complex series of events termed “vascular remodeling,” which includes proliferation of resident smooth muscle cells in the walls of small muscular pulmonary arteries. This process leads to increased thickness of the smooth muscle component of the vessel wall, which then contributes to reduced lumen diameter and increased contractility. Since smooth muscle cell proliferation contributes significantly to the process of vascular remodeling, it is important to appreciate the main factors driving this process in the hypertensive lung. This chapter will review the basic mechanisms of vascular smooth muscle cell proliferation, focusing on work in pulmonary vascular cells.

Nuclear and cytoplasmic scaffold proteins have been shown to be essential for temporal and spatial organization, as well as the fidelity, of MAPK signaling pathways. In this study we show that nesprin-2 is a novel extracellular signal-regulated MAPK1 and 2 (ERK1/2) scaffold protein that serves to regulate nuclear signaling by tethering these kinases at promyelocytic leukemia protein nuclear bodies (PML NBs). Using immunofluorescence microscopy, GST pull-down and immunoprecipitation, we show that nesprin-2, ERK1/2, and PML colocalize and bind to form a nuclear complex. Interference of nesprin-2 function, by either siRNA-mediated knockdown or overexpression of a dominant negative nesprin-2 fragment, augmented ERK1/2 nuclear signaling shown by increased SP1 activity and ELK1 phosphorylation. The functional outcome of nesprin-2 disruption and the resultant sustained ERK1/2 signal was increased proliferation. Importantly, these activities were not induced by previously identified nuclear envelope (NE)-targeted nesprin-2 isoforms but rather were mediated by novel nuclear isoforms that lacked the KASH domain. Taken together, this study suggests that nesprin-2 is a novel intranuclear scaffold, essential for nuclear ERK1/2 signaling fidelity and cell cycle progression.

Introduction BMP4 signals via the Smad pathway to induce the expression of the ID dominant-negative basic helix-loop-helix transcription factors (ID1–4) that regulate cell differentiation. We have shown that ID induction is blunted in human pulmonary artery smooth muscle cells (HPASMCs) from pulmonary arterial hypertension (PAH) patients with mutations in the bone morphogenetic type-II receptor (BMPR-II). TGFβ1 is implicated in the pathogenesis of PAH. We therefore examined whether TGFβ1 and BMP4 signalling directly interact in HPASMCs. Methods Explant-derived HPASMCs from unaffected donors or PAH patients with identified BMPR-II mutations were studied. The transcriptional responses of ID1, ID2, PAI1 and CTGF to BMP4, TGFβ1 or co-treatment were examined by qPCR In some experiments, cells were pre-incubated with cycloheximide or pharmacological inhibitors of ALK5 (SD208), MAP kinase (U0126), TAK1 or p38 MAP kinase (SB203580). The roles of Smad2, Smad3, Smad6 and Smad7 were investigated using siRNAs. Smad-dependent transcription was examined using the BMP-responsive luciferase reporter (BRE-luc) and the TGF-responsive luciferase reporter, CAGA12-luc. Protein lysates collected at 1 and 4hrs were immunoblotted for phosphorylated and total Smads and candidate kinases. In some experiments, nuclear and cytoplasmic fractions were prepared and immunoblotted. Results BMP4 induced ID1 and ID2 expression at 1, 4 and 24h whereas TGFβ1 induced ID1/2 at 1h and repressed them at 4h and 24h. TGFβ1, but not BMP4, induced PAI1 and CTGF expression. BMP4 did not alter TGFβ1-mediated transcriptional responses. In contrast, TGFβ1 attenuated BMP4-mediated ID1/2 induction and the BRE-luc response in donor HPASMCs. Moreover, TGFβ1 abolished BMP4 responses in PAH PASMCs with BMPR-II mutations. This was reversed by the ALK5 inhibitor, SD208, but not by cycloheximide or a TAK1 inhibitor. BMP-Smad phosphorylation and nuclear translocation did not differ between co-treatment and treatment with individual ligands. Smad3 siRNA, but not Smad2, Smad6 or Smad7, reversed the inhibitory effect of TGFβ1. Conclusions TGFβ1 negatively regulates BMP4-mediated ID genes transcription by interfering with BMP-Smad signalling via Smad3, abolishing the BMP signals in cells with BMPR-II mutations. These data reinforce the notion that TGFβ1 plays a pathogenic role in PAH, via direct inhibition of the attenuated BMP response caused by BMPR-II insufficiency.