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Background: Seventy percent of epileptic patients may not experience seizures if they receive proper treatment with antiepileptic drugs (AEDs). However, many children and adolescents face poor seizure control (PSC). Therefore, the purpose of this review is to systematically and quantitatively summarize the pooled prevalence of PSC and its predictors among children with epilepsy in Ethiopia., Methods: The following databases were used to conduct a thorough literature search: Africa Journal of the Online Library, Hinari, Google Scholar, PubMed, Science Direct, EMBASE, Cochrane Database, Sci-Hub, and Scopus. To evaluate the quality of the studies, Newcastle‒Ottawa Scale (NOS) checklists were utilized. The data on study characteristics and prevalence estimates were then combined via random effects meta-analysis, followed by subgroup and sensitivity analyses. Both visual and statistical analyses were employed to assess for any potential publication bias., Results: This review analyzed a total of 12 main studies and reported that the overall prevalence of PSC was 42.42% (95% CI: 33.42-51.42). However, there was significant heterogeneity in the results based on the study region, design, and seizure-free period. The subgroup analysis revealed that the highest prevalence of PSC was found in Southern Nations, Nationalities, and Peoples' (SNNPs) studies (61.88%; 95% CI: 35.91-87.85), studies with a cross-sectional design (46.73%; 95% CI: 36.83-56.62), and studies with a seizure-free period < 6 months (44.69%; 95% CI: 34.51-54.86). However, the lowest prevalence was observed in the Amhara region (35.54%; 95% CI: 27.40-43.67), cohort studies (29.53%; 95% CI: 21.26-38.21), and studies with a seizure-free duration of six months or more (41.64%; 95% CI: 29.94-53.35). The results also revealed a significant correlation between PSC and medication nonadherence (4.64, 95% CI: 2.84-7.58), comorbidities (2.08, 95% CI: 1.01-4.26), and seizure type (3.59, 95% CI: 1.18-10.8)., Conclusion: Based on this review, the findings suggest a notable prevalence of poorly controlled seizures (PSC) among children with epilepsy who are receiving antiepileptic drugs (AEDs) in Ethiopian outpatient epilepsy clinics. Seizures of tonic‒clonic status, comorbidities, and medication nonadherence were associated with poor seizure control., (© 2024. The Author(s).)

Background: Proper disposal of unwanted medicines, in addition to reducing wastage, has a positive impact on the environment and public health. Improper disposal of medications increases the risk of accidental poisonings, particularly among children. This study aimed to assess the level of knowledge, attitudes, and awareness regarding the proper disposal of unused medicines in Gondar city, northwest Ethiopia., Method: From 30 July to 30 August 2023, a community-based cross-sectional study was conducted among the public in Gondar city. The data were gathered using the Kobo toolbox, exported into an Excel sheet, and then analyzed using SPSS version 27. Multivariate and bivariate binary logistic regressions were performed. A p -value of 0.05 with a 95% confidence interval (CI) was used to determine statistical significance., Result: From 786 study participants, the overall knowledge and attitude of the community toward the proper disposal of unused medicines were 42.6 and 42.9%, respectively. Factors identified in this study included ages between 19 and 25 [AOR = 6.91, 95% CI: (3.45, 13.84); education level: secondary [AOR = 11.82, 95% CI: (1.01, 3.29)] and college and above [AOR = 5.68, 95% CI: (2.25, 14.30)]; prior information [AOR = 6.41; 95% CI: (4.02, 10.22)]; and good attitudes [AOR = 2.11; 95% CI: (1.47, 3.02)]] as factors associated with good knowledge toward proper disposal of unused medicines. In addition, receiving information [AOR = 1.86 95% CI: (1.22, 2.86)], taking medication in the past 6 months [AOR = 1.61, 95% CI: (1.09, 2.38)], and being knowledgeable [AOR = 2.07 95% CI: (1.46, 2.94)] were factors contributing to positive attitudes toward the disposal of unused medicines among the general public. Furthermore, approximately 369 participants (46.9%) in our study lacked awareness about the harmful effects of disposing of unused medicine in regular waste., Conclusion: A relatively low level of knowledge and attitudes about the proper disposal of unused medications is present in the community. In this regard, a well-coordinated and methodical public awareness campaign is recommended to disseminate information and promote the appropriate disposal of unused medications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Tegegne, Genet, Workie Limenh, Yohannes, Mohammed Seid, Alemayehu, Ayenew and Simegn.)

Background: Distinct endothelial cell cycle states (early G1 versus late G1) provide different "windows of opportunity" to enable the differential expression of genes that regulate venous versus arterial specification, respectively. Endothelial cell cycle control and arteriovenous identities are disrupted in vascular malformations including arteriovenous shunts, the hallmark of hereditary hemorrhagic telangiectasia (HHT). To date, the mechanistic link between endothelial cell cycle regulation and the development of arteriovenous malformations (AVMs) in HHT is not known., Methods: We used BMP (bone morphogenetic protein) 9/10 blocking antibodies and endothelial-specific deletion of activin A receptor like type 1 ( Alk1 ) to induce HHT in Fucci (fluorescent ubiquitination-based cell cycle indicator) 2 mice to assess endothelial cell cycle states in AVMs. We also assessed the therapeutic potential of inducing endothelial cell cycle G1 state in HHT to prevent AVMs by repurposing the Food and Drug Administration-approved CDK (cyclin-dependent kinase) 4/6 inhibitor (CDK4/6i) palbociclib., Results: We found that endothelial cell cycle state and associated gene expressions are dysregulated during the pathogenesis of vascular malformations in HHT. We also showed that palbociclib treatment prevented AVM development induced by BMP9/10 inhibition and Alk1 genetic deletion. Mechanistically, endothelial cell late G1 state induced by palbociclib modulates the expression of genes regulating arteriovenous identity, endothelial cell migration, metabolism, and VEGF-A (vascular endothelial growth factor A) and BMP9 signaling that collectively contribute to the prevention of vascular malformations., Conclusions: This study provides new insights into molecular mechanisms leading to HHT by defining how endothelial cell cycle is dysregulated in AVMs because of BMP9/10 and Alk1 signaling deficiencies, and how restoration of endothelial cell cycle control may be used to treat AVMs in patients with HHT., Competing Interests: None.

Embryonic definitive hematopoiesis generates hematopoietic stem and progenitor cells (HSPCs) that are essential for the establishment and maintenance of the adult blood system. This process requires the specification of a subset of vascular endothelial cells (ECs) to become hemogenic ECs and to have subsequent endothelial-to-hematopoietic transition (EHT), and the underlying mechanisms are largely undefined. We identified microRNA (miR)-223 as a negative regulator of murine hemogenic EC specification and EHT. Loss of miR-223 leads to increased formation of hemogenic ECs and HSPCs, which is associated with increased retinoic acid signaling, which we previously showed as promoting hemogenic EC specification. Additionally, loss of miR-223 leads to the generation of myeloid-biased hemogenic ECs and HSPCs, which results in an increased proportion of myeloid cells throughout embryonic and postnatal life. Our findings identify a negative regulator of hemogenic EC specification and highlight the importance of this process for the establishment of the adult blood system., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Connexin37-mediated regulation of cell cycle modulators and, consequently, growth arrest lack mechanistic understanding. We previously showed that arterial shear stress up-regulates Cx37 in endothelial cells and activates a Notch/Cx37/p27 signaling axis to promote G1 cell cycle arrest, and this is required to enable arterial gene expression. However, how induced expression of a gap junction protein, Cx37, up-regulates cyclin-dependent kinase inhibitor p27 to enable endothelial growth suppression and arterial specification is unclear. Herein, we fill this knowledge gap by expressing wild-type and regulatory domain mutants of Cx37 in cultured endothelial cells expressing the Fucci cell cycle reporter. We determined that both the channel-forming and cytoplasmic tail domains of Cx37 are required for p27 up-regulation and late G1 arrest. Mechanistically, the cytoplasmic tail domain of Cx37 interacts with, and sequesters, activated ERK in the cytoplasm. This then stabilizes pERK nuclear target Foxo3a, which up-regulates p27 transcription. Consistent with previous studies, we found this Cx37/pERK/Foxo3a/p27 signaling axis functions downstream of arterial shear stress to promote endothelial late G1 state and enable up-regulation of arterial genes., (© 2023 Acharya et al.)

The subventricular zone (SVZ) is the largest neural stem cell (NSC) niche in the adult brain; herein, the blood-brain barrier is leaky, allowing direct interactions between NSCs and endothelial cells (ECs). Mechanisms by which direct NSC-EC interactions in the adult SVZ control NSC behavior are unclear. We found that Cx43 is highly expressed by SVZ NSCs and ECs, and its deletion in either leads to increased NSC proliferation and neuroblast generation, suggesting that Cx43-mediated NSC-EC interactions maintain NSC quiescence. This is further supported by single-cell RNA sequencing and in vitro studies showing that ECs control NSC proliferation by regulating expression of genes associated with NSC quiescence and/or activation in a Cx43-dependent manner. Cx43 mediates these effects in a channel-independent manner involving its cytoplasmic tail and ERK activation. Such insights inform adult NSC regulation and maintenance aimed at stem cell therapies for neurodegenerative disorders., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The rod-shaped adult cardiomyocyte (CM) harbors a unique architecture of its lateral surface with periodic crests, relying on the presence of subsarcolemmal mitochondria (SSM) with unknown role. Here, we investigated the development and functional role of CM crests during the postnatal period. We found in rodents that CM crest maturation occurs late between postnatal day 20 (P20) and P60 through both SSM biogenesis, swelling and crest-crest lateral interactions between adjacent CM, promoting tissue compaction. At the functional level, we showed that the P20-P60 period is dedicated to the improvement of relaxation. Interestingly, crest maturation specifically contributes to an atypical CM hypertrophy of its short axis, without myofibril addition, but relying on CM lateral stretching. Mechanistically, using constitutive and conditional CM-specific knock-out mice, we identified ephrin-B1, a lateral membrane stabilizer, as a molecular determinant of P20-P60 crest maturation, governing both the CM lateral stretch and the diastolic function, thus highly suggesting a link between crest maturity and diastole. Remarkably, while young adult CM-specific Efnb1 KO mice essentially exhibit an impairment of the ventricular diastole with preserved ejection fraction and exercise intolerance, they progressively switch toward systolic heart failure with 100% KO mice dying after 13 months, indicative of a critical role of CM-ephrin-B1 in the adult heart function. This study highlights the molecular determinants and the biological implication of a new late P20-P60 postnatal developmental stage of the heart in rodents during which, in part, ephrin-B1 specifically regulates the maturation of the CM surface crests and of the diastolic function., Competing Interests: CK, CG, GG, MS, PA, OC, AM, YB, BT, MC, SR, SA, RF, NP, RM, PS, AD, TC, JS, CG No competing interests declared, CD, JM, TS Dubroca, Julie Maupoint and Thierry Sulpice are employees of Cardiomedex, (© 2023, Karsenty et al.)

During blood vessel development, endothelial cells become specified toward arterial or venous fates to generate a circulatory network that provides nutrients and oxygen to, and removes metabolic waste from, all tissues. Arterial-venous specification occurs in conjunction with suppression of endothelial cell cycle progression; however, the mechanistic role of cell cycle state is unknown. Herein, using Cdh5-CreER T2 ;R26FUCCI2aR reporter mice, we find that venous endothelial cells are enriched for the FUCCI-Negative state (early G1) and BMP signaling, while arterial endothelial cells are enriched for the FUCCI-Red state (late G1) and TGF-β signaling. Furthermore, early G1 state is essential for BMP4-induced venous gene expression, whereas late G1 state is essential for TGF-β1-induced arterial gene expression. Pharmacologically induced cell cycle arrest prevents arterial-venous specification defects in mice with endothelial hyperproliferation. Collectively, our results show that distinct endothelial cell cycle states provide distinct windows of opportunity for the molecular induction of arterial vs. venous fate., (© 2022. The Author(s).)

Diabetes mellitus is a serious, chronic disease that occurs either when the pancreas does not produce enough insulin, or when the body can't effectively use insulin. Herbal medicines have been commonly used by diabetic patients for the treatment of diabetes mellitus. To include findings from different studies, publications related to in vivo and invitro antidiabetic activities of medicinal plants in Ethiopia were searched from different databases, such as Web of Science, Google Scholar, Medline, Scopus, and PubMed, using English key terms. Different medicinal plant parts were used experimentally for antidiabetic effects in Ethiopia. Among these, leaves (69%) were the most commonly investigated medicinal plant parts followed by roots (14%) and seeds (7%). Most of the investigations were completed with hydro-methanolic extracts to obtain a higher percentage of yield. Medicinal plants such as Thymus schemperi R , Thymus vulgaris L , Hagenia abyssinica, Aloe megalacantha baker, Aloe moticola Reyonolds, Aloe pulecherrima Gilbert & sebseb , Bersama abyssinica fresen , and Rubus Erlangeri Engl have shown in vitro α-amylase inhibitory activity. However, only Hagenia abyssinica, Thymus schemperi R , and Thymus vulgaris L have exhibited α-glucosidase inhibitory activity. Likewise, only the extract of Aloe pulecherrima Gilbert & sebseb posses' maltase and sucrose inhibitory activity. In vivo antidiabetic activity were conducted for the extract of medicinal plants such as A. remota, S. rebaudiani, T. schemperi, T. vulgaris, H. abyssinica, C. aurea, D. stramonium, A. megalacantha, A. moticola, A.integrifolia, A. pulecherrima, B. grandiflorum, B. abyssinica, P. schimperiana, M. stenopetala, C. aure, J. schimperiana, T. brownie, C. macrostachys, I. spicata, O. integrifolia, C. abyssinica, R. Erlangeri, L. culinaris, A. camperi, A. polystachyus, A. ilicifalius, C. tomentosa, and C. Edulis. This review gives collective evidence on the potential antidiabetic activities of medicinal plants in Ethiopia. Moreover, further studies are recommended to substantiate the use of these medicinal plants as an antidiabetic agent., Competing Interests: The authors declare that they have no competing interests., (© 2022 The Authors.)

Endothelial cells (ECs) comprise the lumenal lining of all blood vessels and are critical for the functioning of the cardiovascular system. Their phenotypes can be modulated by alternative splicing of RNA to produce distinct protein isoforms. To characterize the RNA and protein isoform landscape within ECs, we applied a long read proteogenomics approach to analyse human umbilical vein endothelial cells (HUVECs). Transcripts delineated from PacBio sequencing serve as the basis for a sample-specific protein database used for downstream mass-spectrometry (MS) analysis to infer protein isoform expression. We detected 53,863 transcript isoforms from 10,426 genes, with 22,195 of those transcripts being novel. Furthermore, the predominant isoform in HUVECs does not correspond with the accepted "reference isoform" 25% of the time, with vascular pathway-related genes among this group. We found 2,597 protein isoforms supported through unique peptides, with an additional 2,280 isoforms nominated upon incorporation of long-read transcript evidence. We characterized a novel alternative acceptor for endothelial-related gene CDH5, suggesting potential changes in its associated signalling pathways. Finally, we identified novel protein isoforms arising from a diversity of RNA splicing mechanisms supported by uniquely mapped novel peptides. Our results represent a high-resolution atlas of known and novel isoforms of potential relevance to endothelial phenotypes and function.[Figure: see text].

There are two vascular networks in mammals that coordinately function as the main supply and drainage systems of the body. The blood vasculature carries oxygen, nutrients, circulating cells, and soluble factors to and from every tissue. The lymphatic vasculature maintains interstitial fluid homeostasis, transports hematopoietic cells for immune surveillance, and absorbs fat from the gastrointestinal tract. These vascular systems consist of highly organized networks of specialized vessels including arteries, veins, capillaries, and lymphatic vessels that exhibit different structures and cellular composition enabling distinct functions. All vessels are composed of an inner layer of endothelial cells that are in direct contact with the circulating fluid; therefore, they are the first responders to circulating factors. However, endothelial cells are not homogenous; rather, they are a heterogenous population of specialized cells perfectly designed for the physiological demands of the vessel they constitute. This review provides an overview of the current knowledge of the specification of arterial, venous, capillary, and lymphatic endothelial cell identities during vascular development. We also discuss how the dysregulation of these processes can lead to vascular malformations, and therapeutic approaches that have been developed for their treatment.

Objective: To evaluate recurrence and early postoperative complications (sepsis) following surgical excision combined with radiotherapy for troublesome hip heterotopic ossification in patients with spinal cord injury and traumatic brain injury., Design: Retrospective case-control study., Setting: Data relating to patients with spinal cord injury or traumatic brain injury who underwent surgical excision of hip heterotopic ossification were retrieved from the BANKHO database. Case patients underwent excision + radiotherapy and controls underwent excision only. Control patients were matched to case patients according to sex and age (± 4 years)., Participants: Data from 19 case patients and 76 controls were analysed., Interventions: Not applicable., Main Outcome Measure: The primary end-point was recurrence of heterotopic ossification. Secondary end-points were postoperative complications and, more specifically, sepsis that required surgical revision., Results: There was no difference between the odds ratios (OR) for recurrence for each group (OR case group = 0.63, OR spinal cord injury subgroup = 0.45 and OR head injury subgroup = 1.04). The rate of sepsis requiring surgical revision was significantly higher in the case group (p < 0.05)., Conclusion: Based on the results of this case-control study, we suggest that radiotherapy should not be combined with surgery in patients with troublesome hip heterotopic ossification undergoing excision. Radiotherapy does not appear to prevent recurrence and, moreover, it is associated with an increased risk of postoperative sepsis.

Endothelial cell migration, proliferation and survival are triggered by VEGF-A activation of VEGFR2. However, how these cell behaviors are regulated individually is still unknown. Here we identify Endophilin-A2 (ENDOA2), a BAR-domain protein that orchestrates CLATHRIN-independent internalization, as a critical mediator of endothelial cell migration and sprouting angiogenesis. We show that EndoA2 knockout mice exhibit postnatal angiogenesis defects and impaired front-rear polarization of sprouting tip cells. ENDOA2 deficiency reduces VEGFR2 internalization and inhibits downstream activation of the signaling effector PAK but not ERK, thereby affecting front-rear polarity and migration but not proliferation or survival. Mechanistically, VEGFR2 is directed towards ENDOA2-mediated endocytosis by the SLIT2-ROBO pathway via SLIT-ROBO-GAP1 bridging of ENDOA2 and ROBO1. Blocking ENDOA2-mediated endothelial cell migration attenuates pathological angiogenesis in oxygen-induced retinopathy models. This work identifies a specific endocytic pathway controlling a subset of VEGFR2 mediated responses that could be targeted to prevent excessive sprouting angiogenesis in pathological conditions.

Background: Hereditary hemorrhagic telangiectasia (HHT) is an inherited vascular disorder that causes arteriovenous malformations (AVMs). Mutations in the genes encoding Endoglin ( ENG) and activin-receptor-like kinase 1 ( AVCRL1 encoding ALK1) cause HHT type 1 and 2, respectively. Mutations in the SMAD4 gene are present in families with juvenile polyposis-HHT syndrome that involves AVMs. SMAD4 is a downstream effector of transforming growth factor-β (TGFβ)/bone morphogenetic protein (BMP) family ligands that signal via activin-like kinase receptors (ALKs). Ligand-neutralizing antibodies or inducible, endothelial-specific Alk1 deletion induce AVMs in mouse models as a result of increased PI3K (phosphatidylinositol 3-kinase)/AKT (protein kinase B) signaling. Here we addressed if SMAD4 was required for BMP9-ALK1 effects on PI3K/AKT pathway activation., Methods: The authors generated tamoxifen-inducible, postnatal, endothelial-specific Smad4 mutant mice ( Smad4 iΔEC )., Results: We found that loss of endothelial Smad4 resulted in AVM formation and lethality. AVMs formed in regions with high blood flow in developing retinas and other tissues. Mechanistically, BMP9 signaling antagonized flow-induced AKT activation in an ALK1- and SMAD4-dependent manner. Smad4 iΔEC endothelial cells in AVMs displayed increased PI3K/AKT signaling, and pharmacological PI3K inhibitors or endothelial Akt1 deletion both rescued AVM formation in Smad4 iΔEC mice. BMP9-induced SMAD4 inhibited casein kinase 2 ( CK2) transcription, in turn limiting PTEN phosphorylation and AKT activation. Consequently, CK2 inhibition prevented AVM formation in Smad4 iΔEC mice., Conclusions: Our study reveals SMAD4 as an essential effector of BMP9-10/ALK1 signaling that affects AVM pathogenesis via regulation of CK2 expression and PI3K/AKT1 activation.

Excess dietary lipid uptake causes obesity, a major global health problem. Enterocyte-absorbed lipids are packaged into chylomicrons, which enter the bloodstream through intestinal lymphatic vessels called lacteals. Here, we show that preventing lacteal chylomicron uptake by inducible endothelial genetic deletion of Neuropilin1 ( Nrp1 ) and Vascular endothelial growth factor receptor 1 ( Vegfr1 ; also known as Flt1 ) renders mice resistant to diet-induced obesity. Absence of NRP1 and FLT1 receptors increased VEGF-A bioavailability and signaling through VEGFR2, inducing lacteal junction zippering and chylomicron malabsorption. Restoring permeable lacteal junctions by VEGFR2 and vascular endothelial (VE)-cadherin signaling inhibition rescued chylomicron transport in the mutant mice. Zippering of lacteal junctions by disassembly of cytoskeletal VE-cadherin anchors prevented chylomicron uptake in wild-type mice. These data suggest that lacteal junctions may be targets for preventing dietary fat uptake., (Copyright © 2018, American Association for the Advancement of Science.)

Neurogenic heterotopic ossification (NHO) is the formation of ectopic bone generally in muscles surrounding joints following spinal cord or brain injury. We investigated the mechanisms of NHO formation in 64 patients and a mouse model of spinal cord injury-induced NHO. We show that marrow from human NHOs contains hematopoietic stem cell (HSC) niches, in which mesenchymal stromal cells (MSCs) and endothelial cells provide an environment supporting HSC maintenance, proliferation, and differentiation. The transcriptomic signature of MSCs from NHOs shows a neuronal imprinting associated with a molecular network required for HSC support. We demonstrate that oncostatin M (OSM) produced by activated macrophages promotes osteoblastic differentiation and mineralization of human muscle-derived stromal cells surrounding NHOs. The key role of OSM was confirmed using an experimental model of NHO in mice defective for the OSM receptor (OSMR). Our results provide strong evidence that macrophages contribute to NHO formation through the osteogenic action of OSM on muscle cells within an inflammatory context and suggest that OSM/OSMR could be a suitable therapeutic target. Altogether, the evidence of HSCs in ectopic bones growing at the expense of soft tissue in spinal cord/brain-injured patients indicates that inflammation and muscle contribute to HSC regulation by the brain-bone-blood triad.

Activin receptor-like kinase 1 (ALK1) is an endothelial serine-threonine kinase receptor for bone morphogenetic proteins (BMPs) 9 and 10. Inactivating mutations in the ALK1 gene cause hereditary haemorrhagic telangiectasia type 2 (HHT2), a disabling disease characterized by excessive angiogenesis with arteriovenous malformations (AVMs). Here we show that inducible, endothelial-specific homozygous Alk1 inactivation and BMP9/10 ligand blockade both lead to AVM formation in postnatal retinal vessels and internal organs including the gastrointestinal (GI) tract in mice. VEGF and PI3K/AKT signalling are increased on Alk1 deletion and BMP9/10 ligand blockade. Genetic deletion of the signal-transducing Vegfr2 receptor prevents excessive angiogenesis but does not fully revert AVM formation. In contrast, pharmacological PI3K inhibition efficiently prevents AVM formation and reverts established AVMs. Thus, Alk1 deletion leads to increased endothelial PI3K pathway activation that may be a novel target for the treatment of vascular lesions in HHT2.

Vascular permeability and neovascularization are implicated in many diseases including retinopathies and diabetic wound healing. Robo4 is an endothelial-specific transmembrane receptor that stabilizes the vasculature, as shown in Robo4 -/- mice that develop hyperpermeability, but how Robo4 signals remained unclear. Here we show that Robo4 deletion enhances permeability and revascularization in oxygen-induced retinopathy (OIR) and accelerates cutaneous wound healing. To determine Robo4 signalling pathways, we generated transgenic mice expressing a truncated Robo4 lacking the cytoplasmic domain (Robo4ΔCD). Robo4ΔCD expression is sufficient to prevent permeability, and inhibits OIR revascularization and wound healing in Robo4 -/- mice. Mechanistically, Robo4 does not affect Slit2 signalling, but Robo4 and Robo4ΔCD counteract Vegfr2-Y949 (Y951 in human VEGFR2) phosphorylation by signalling through the endothelial UNC5B receptor. We conclude that Robo4 inhibits angiogenesis and vessel permeability independently of its cytoplasmic domain, while activating VEGFR2-Y951 via ROBO4 inhibition might accelerate tissue revascularization in retinopathy of prematurity and in diabetic patients.

Background: Sprouting angiogenesis is a key process driving blood vessel growth in ischemic tissues and an important drug target in a number of diseases, including wet macular degeneration and wound healing. Endothelial cells forming the sprout must develop front-rear polarity to allow sprout extension. The adaptor proteins Nck1 and 2 are known regulators of cytoskeletal dynamics and polarity, but their function in angiogenesis is poorly understood. Here, we show that the Nck adaptors are required for endothelial cell front-rear polarity and migration downstream of the angiogenic growth factors VEGF-A and Slit2., Methods and Results: Mice carrying inducible, endothelial-specific Nck1/2 deletions fail to develop front-rear polarized vessel sprouts and exhibit severe angiogenesis defects in the postnatal retina and during embryonic development. Inactivation of NCK1 and 2 inhibits polarity by preventing Cdc42 and Pak2 activation by VEGF-A and Slit2. Mechanistically, NCK binding to ROBO1 is required for both Slit2- and VEGF-induced front-rear polarity. Selective inhibition of polarized endothelial cell migration by targeting Nck1/2 prevents hypersprouting induced by Notch or Bmp signaling inhibition, and pathological ocular neovascularization and wound healing, as well., Conclusions: These data reveal a novel signal integration mechanism involving NCK1/2, ROBO1/2, and VEGFR2 that controls endothelial cell front-rear polarity during sprouting angiogenesis., (© 2015 American Heart Association, Inc.)

Ocular neovascular diseases are a leading cause of blindness. Vascular endothelial growth factor (VEGF) blockade improves vision, but not all individuals respond to anti-VEGF treatment, making additional means to prevent neovascularization necessary. Slit-family proteins (Slits) are ligands of Roundabout (Robo) receptors that repel developing axons in the nervous system. Robo1 expression is altered in ocular neovascular diseases, and previous in vitro studies have reported both pro- and anti-angiogenic effects of Slits. However, genetic evidence supporting a role for Slits in ocular neovascularization is lacking. Here we generated conditional knockout mice deficient in various Slit and Robo proteins and found that Slit2 potently and selectively promoted angiogenesis via Robo1 and Robo2 in mouse postnatal retina and in a model of ocular neovascular disease. Mechanistically, Slit2 acting through Robo1 and Robo2 promoted the migration of endothelial cells. These receptors are required for both Slit2- and VEGF-induced Rac1 activation and lamellipodia formation. Thus, Slit2 blockade could potentially be used therapeutically to inhibit angiogenesis in individuals with ocular neovascular disease.

Loss of T-tubules (TT), sarcolemmal invaginations of cardiomyocytes (CMs), was recently identified as a general heart failure (HF) hallmark. However, whether TT per se or the overall sarcolemma is altered during HF process is still unknown. In this study, we directly examined sarcolemmal surface topography and physical properties using Atomic Force Microscopy (AFM) in living CMs from healthy and failing mice hearts. We confirmed the presence of highly organized crests and hollows along myofilaments in isolated healthy CMs. Sarcolemma topography was tightly correlated with elasticity, with crests stiffer than hollows and related to the presence of few packed subsarcolemmal mitochondria (SSM) as evidenced by electron microscopy. Three days after myocardial infarction (MI), CMs already exhibit an overall sarcolemma disorganization with general loss of crests topography thus becoming smooth and correlating with a decreased elasticity while interfibrillar mitochondria (IFM), myofilaments alignment and TT network were unaltered. End-stage post-ischemic condition (15days post-MI) exacerbates overall sarcolemma disorganization with, in addition to general loss of crest/hollow periodicity, a significant increase of cell surface stiffness. Strikingly, electron microscopy revealed the total depletion of SSM while some IFM heaps could be visualized beneath the membrane. Accordingly, mitochondrial Ca(2+) studies showed a heterogeneous pattern between SSM and IFM in healthy CMs which disappeared in HF. In vitro, formamide-induced sarcolemmal stress on healthy CMs phenocopied post-ischemic kinetics abnormalities and revealed initial SSM death and crest/hollow disorganization followed by IFM later disarray which moved toward the cell surface and structured heaps correlating with TT loss. This study demonstrates that the loss of crest/hollow organization of CM surface in HF occurs early and precedes disruption of the TT network. It also highlights a general stiffness increased of the CM surface most likely related to atypical IFM heaps while SSM died during HF process. Overall, these results indicate that initial sarcolemmal stress leading to SSM death could underlie subsequent TT disarray and HF setting., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Sympathetic nervous system (SNS) plays a key role in cardiac homeostasis and its deregulations always associate with bad clinical outcomes. To date, little is known about molecular mechanisms regulating cardiac sympathetic innervation. The aim of the study was to determine the role of fibroblasts in heart sympathetic innervation. RT-qPCR and western-blots analysis performed in cardiomyocytes and fibroblasts isolated from healthy adult rat hearts revealed that Pro-Nerve growth factor (NGF) and pro-differentiating mature NGF were the most abundant neurotrophins expressed in cardiac fibroblasts while barely detectable in cardiomyocytes. When cultured with cardiac fibroblasts or fibroblast-conditioned medium, PC12 cells differentiated into/sympathetic-like neurons expressing axonal marker Tau-1 at neurites in contact with cardiomyocytes. This was prevented by anti-NGF blocking antibodies suggesting a paracrine action of NGF secreted by fibroblasts. When co-cultured with cardiomyocytes to mimic neurocardiac synapse, differentiated PC12 cells exhibited enhanced norepinephrine secretion as quantified by HPLC compared to PC12 cultured alone while co-culture with fibroblasts had no effect. However, when supplemented to PC12-cardiomyocytes co-culture, fibroblasts allowed long-term survival of the neurocardiac synapse. Activated fibroblasts (myofibroblasts) isolated from myocardial infarction rat hearts exhibited significantly higher mature NGF expression than normal fibroblasts and also promoted PC12 cells differentiation. Within the ischemic area lacking cardiomyocytes and neurocardiac synapses, tyrosine hydroxylase immunoreactivity was increased and associated with local anarchical and immature sympathetic hyperinnervation but tissue norepinephrine content was similar to that of normal cardiac tissue, suggesting depressed sympathetic function. Collectively, these findings demonstrate for the first time that fibroblasts are essential for the setting of cardiac sympathetic innervation and neurocardiac synapse stability. They also suggest that neurocardiac synapse functionality relies on a triptych with tight interaction between sympathetic nerve endings, cardiomyocytes and fibroblasts. Deregulations of this triptych may be involved in pathophysiology of cardiac diseases.

All cardiomyopathies and more specifically myocardial infarction always evolve to cardiomyocytes death and the ensuing heart failure setting. So far, cardiac regenerative medicine has focused on the use of stem cells and completely ignored the resident cardiomyocytes, assumed in a postmitotic state. However, recent findings in zebrafish and mammalians challenge this view and suggest that these cells have some capacity to proliferate and can contribute to heart regeneration. In this review, we propose an overall synthesis about knowledge of the proliferative and regenerative capacities of resident cardiomyocytes, dealing with some mechanistic aspects. In the future, the accurate identification of molecular mechanisms allowing wake-up of resident cardiomyocyte proliferation will certainly open new therapeutic avenues in cardiac regeneration., (© 2012 médecine/sciences – Inserm / SRMS.)

Objectives: Cardiac surgery with cardiopulmonary bypass (CPB) causes severe derangements in the hemostatic system, which in turn puts the patient at risks of microvascular bleeding. Excessive transfusion and surgical re-exploration after cardiac surgery are potentially associated with a number of adverse outcomes including increased mortality., Design: This review describes coagulopathies occurring in cardiac surgery and the monitoring of these. Viscoelastic hemostatic assays (VHA) have routinely been used in cardiac surgery for more than 25 years and at least 16 studies (involving more than 3250 patients, including three randomized controlled trials) have compared VHA to routine coagulation tests (RCoT) with regards to their ability to predict bleeding and re-do surgery and also with regards to transfusion requirements secondary to hemostatic therapy based on VHA vs. RCoT., Results: All 16 studies demonstrated superiority with VHA as compared to RCoT both in predicting bleeding and need for re-do surgery and in reducing the total amount of blood transfusions by employing goal-directed administration of blood products based on VHA as compared RCoT based therapy or at the clinicians discretion., Conclusions: The different types of coagulopathies observed in patients undergoing cardiac surgery requires adequate and timely hemostatic therapy and real-time monitoring with WHA such as TEG/ROTEM and whole blood platelet aggregometry.

Rationale: Cardiac tissue cohesion relying on highly ordered cardiomyocytes (CM) interactions is critical because most cardiomyopathies are associated with tissue remodeling and architecture alterations., Objective: Eph/ephrin system constitutes a ubiquitous system coordinating cellular communications which recently emerged as a major regulator in adult organs. We examined if eph/ephrin could participate in cardiac tissue cyto-organization., Methods and Results: We reported the expression of cardiac ephrin-B1 in both endothelial cells and for the first time in CMs where ephrin-B1 localized specifically at the lateral membrane. Ephrin-B1 knock-out (KO) mice progressively developed cardiac tissue disorganization with loss of adult CM rod-shape and sarcomeric and intercalated disk structural disorganization confirmed in CM-specific ephrin-B1 KO mice. CMs lateral membrane exhibited abnormal structure by electron microscopy and notably increased stiffness by atomic force microscopy. In wild-type CMs, ephrin-B1 interacted with claudin-5/ZO-1 complex at the lateral membrane, whereas the complex disappeared in KO/CM-specific ephrin-B1 KO mice. Ephrin-B1 deficiency resulted in decreased mRNA expression of CM basement membrane components and disorganized fibrillar collagen matrix, independently of classical integrin/dystroglycan system. KO/CM-specific ephrin-B1 KO mice exhibited increased left ventricle diameter and delayed atrioventricular conduction. Under pressure overload stress, KO mice were prone to death and exhibited striking tissue disorganization. Finally, failing CMs displayed downregulated ephrin-B1/claudin-5 gene expression linearly related to the ejection fraction., Conclusions: Ephrin-B1 is necessary for cardiac tissue architecture cohesion by stabilizing the adult CM morphology through regulation of its lateral membrane. Because decreased ephrin-B1 is associated with molecular/functional cardiac defects, it could represent a new actor in the transition toward heart failure.