Coronary Artery Disease Risk Variant Dampens the Expression of CALCRL by Reducing HSF Binding to Shear Stress Responsive Enhancer in Endothelial Cells In Vitro.

BACKGROUND: CALCRL (calcitonin receptor-like) protein is an important mediator of the endothelial fluid shear stress response, which is associated with the genetic risk of coronary artery disease. In this study, we functionally characterized the noncoding regulatory elements carrying coronary artery disease that risks single-nucleotide polymorphisms and studied their role in the regulation of CALCRL expression in endothelial cells. METHODS: To functionally characterize the coronary artery disease single-nucleotide polymorphisms harbored around the gene CALCRL, we applied an integrative approach encompassing statistical, transcriptional (RNA-seq), and epigenetic (ATAC-seq, chromatin immunoprecipitation assay-quantitative polymerase chain reaction, and electromobility shift assay) analyses, alongside luciferase reporter assays, and targeted gene and enhancer perturbations (siRNA and clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) in human aortic endothelial cells. RESULTS: We demonstrate that the regulatory element harboring rs880890 exhibits high enhancer activity and shows significant allelic bias. The A allele was favored over the G allele, particularly under shear stress conditions, mediated through alterations in the HSF1 (heat shock factor 1) motif and binding. CRISPR deletion of rs880890 enhancer resulted in downregulation of CALCRL expression, whereas HSF1 knockdown resulted in a significant decrease in rs880890-enhancer activity and CALCRL expression. A significant decrease in HSF1 binding to the enhancer region in endothelial cells was observed under disturbed flow compared with unidirectional flow. CALCRL knockdown and variant perturbation experiments indicated the role of CALCRL in mediating eNOS (endothelial NO synthase), APLN (apelin), angiopoietin, prostaglandins, and EDN1 (endothelin-1) signaling pathways leading to a decrease in cell proliferation, tube formation, and NO production. CONCLUSIONS: Overall, our results demonstrate the existence of an endothelial-specific HSF (heat shock factor)-regulated transcriptional enhancer that mediates CALCRL expression. A better understanding of CALCRL gene regulation and the role of single-nucleotide polymorphisms in the modulation of CALCRL expression could provide important steps toward understanding the genetic regulation of shear stress signaling responses.

Proteomics on human cerebral cavernous malformations reveals novel biomarkers in neurovascular dysfunction for the disease pathology.

BACKGROUND: Cerebral cavernous malformation (CCM) is a disease associated with an elevated risk of focal neurological deficits, seizures, and hemorrhagic stroke. The disease has an inflammatory profile and improved knowledge of CCM pathology mechanisms and exploration of candidate biomarkers will enable new non-invasive treatments. METHODS: We analyzed protein signatures in human CCM tissue samples by using a highly specific and sensitive multiplexing technique, proximity extension assay. FINDINGS: Data analysis revealed CCM specific proteins involved in endothelial dysfunction/inflammation/activation, leukocyte infiltration/chemotaxis, hemostasis, extracellular matrix dysfunction, astrocyte and microglial cell activation. Biomarker expression profiles matched bleeding status, especially with higher levels of inflammatory markers and activated astrocytes in ruptured than non-ruptured samples, some of these biomarkers are secreted into blood or urine. Furthermore, analysis was also done in a spatially resolving manner by separating the lesion area from the surrounding brain tissue. Our spatial studies revealed that although appearing histologically normal, the CCM border areas were pathological when compared to control brain tissues. Moreover, the functional relevance of CD93, ICAM-1 and MMP9, markers related to endothelial cell activation and extracellular matrix was validated by a murine pre-clinical CCM model. INTERPRETATION: Here we present a novel strategy for proteomics analysis on human CCMs, offering a possibility for high-throughput protein screening acquiring data on the local environment in the brain. Our data presented here describe CCM relevant brain proteins and specifically those which are secreted can serve the need of circulating CCM biomarkers to predict cavernoma's risk of bleeding.

Circulating biomarkers in familial cerebral cavernous malformation.

BACKGROUND: Cerebral Cavernous Malformation (CCM) is a rare cerebrovascular disease, characterized by the presence of multiple vascular malformations that may result in intracerebral hemorrhages (ICHs), seizure(s), or focal neurological deficits (FND). Familial CCM (fCCM) is due to loss of function mutations in one of the three independent genes KRIT1 (CCM1), Malcavernin (CCM2), or Programmed Cell death 10 (PDCD10/CCM3). The aim of this study was to identify plasma protein biomarkers of fCCM to assess the severity of the disease and predict its progression. METHODS: Here, we have investigated plasma samples derived from n = 71 symptomatic fCCM patients (40 female/31 male) and n = 17 healthy donors (HD) (9 female/8 male) of the Phase 1/2 Treat_CCM trial, using multiplexed protein profiling approaches. FINDINGS: Biomarkers as sCD14 (p = 0.00409), LBP (p = 0.02911), CXCL4 (p = 0.038), ICAM-1 (p = 0.02013), ANG2 (p = 0.026), CCL5 (p = 0.00403), THBS1 (p = 0.0043), CRP (p = 0.0092), and HDL (p = 0.027), were significantly different in fCCM compared to HDs. Of note, sENG (p = 0.011), THBS1 (p = 0.011) and CXCL4 (p = 0.011), were correlated to CCM genotype. sROBO4 (p = 0.014), TM (p = 0.026) and CRP (p = 0.040) were able to predict incident adverse clinical events, such as ICH, FND or seizure. GDF-15, FLT3L, CXCL9, FGF-21 and CDCP1, were identified as predictors of the formation of new MRI-detectable lesions over 2-year follow-up. Furthermore, the functional relevance of ang2, thbs1, robo4 and cdcp1 markers was validated by zebrafish pre-clinical model of fCCM. INTERPRETATION: Overall, our study identifies a set of biochemical parameters to predict CCM progression, suggesting biological interpretations and potential therapeutic approaches to CCM disease. FUNDING: Italian Medicines Agency, Associazione Italiana per la Ricerca sul Cancro (AIRC), ERC, Leducq Transatlantic Network of Excellence, Swedish Research Council.

Stromal transdifferentiation drives lipomatosis and induces extensive vascular remodeling in the aging human lymph node.

Lymph node (LN) lipomatosis is a common but rarely discussed phenomenon associated with aging that involves a gradual exchange of the LN parenchyma into adipose tissue. The mechanisms behind these changes and the effects on the LN are unknown. We show that LN lipomatosis starts in the medullary regions of the human LN and link the initiation of lipomatosis to transdifferentiation of LN fibroblasts into adipocytes. The latter is associated with a downregulation of lymphotoxin beta expression. We also show that isolated medullary and CD34+ fibroblasts, in contrast to the reticular cells of the T-cell zone, display an inherently higher sensitivity for adipogenesis. Progression of lipomatosis leads to a gradual loss of the medullary lymphatic network, but at later stages, collecting-like lymphatic vessels are found inside the adipose tissue. The stromal dysregulation includes a dramatic remodeling and dilation of the high endothelial venules associated with reduced density of naïve T-cells. Abnormal clustering of plasma cells is also observed. Thus, LN lipomatosis causes widespread stromal dysfunction with consequences for the immune contexture of the human LN. Our data warrant an increased awareness of LN lipomatosis as a factor contributing to decreased immune functions in the elderly and in disease. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Slow-Closing Clip for the Treatment of Nonsaccular Vertebrobasilar Aneurysms: A Retrospective Case Series.

OBJECTIVE: Vertebrobasilar artery nonsaccular aneurysms (VBANSAs) are associated with a 13% annual mortality. Revascularization and flow diversion are life-saving options in select cases; technical failures and rapid hemodynamic changes may contribute to unwanted outcomes. We describe a technique and report clinical outcomes of patients treated with an experimental slow-closing clip (SCC). METHODS: An experimental SCC was created to gradually close the parent artery of aneurysms. Clinical, radiographic, and outcome data from patients with VBANSAs who underwent experimental treatment with the SCC were retrospectively analyzed. RESULTS: Among 10 patients (7 men; mean age, 49.5 years; range, 18-73 years), 6 presented with mass effect symptoms, 1 with ischemic stroke, 2 with subarachnoid hemorrhage, and 1 with hydrocephalus. Five patients underwent revascularization plus SCC application, and 5 were treated with SCC alone. The mean follow-up was 6.7 years. The expected mortality among patients with unruptured VBANSAs with previous treatment options in this period was 52.7%, whereas the observed rate was 20%. Four patients died within 12 months after treatment. Causes of death were brainstem ischemic stroke, poor-grade subarachnoid hemorrhage, poor clinical presentation, and unknown. Six patients were alive at last follow-up, with unchanged or improved modified Rankin Scale scores. Mortality was associated with posterior-projecting aneurysms and late-stage treatment. CONCLUSIONS: In this small case series, use of SCC overcame the natural history of VBANSAs when treatment timing and aneurysm anatomy were suitable. The SCC potentially favors aneurysm thrombosis and collateral reactivation. More studies are necessary to better develop the SCC.

Immunothrombosis and vascular heterogeneity in cerebral cavernous malformation.

Cerebral cavernous malformation (CCM) is a neurovascular disease that results in various neurological symptoms. Thrombi have been reported in surgically resected CCM patient biopsies, but the molecular signatures of these thrombi remain elusive. Here, we investigated the kinetics of thrombi formation in CCM and how thrombi affect the vasculature and contribute to cerebral hypoxia. We used RNA sequencing to investigate the transcriptome of mouse brain endothelial cells with an inducible endothelial-specific Ccm3 knock-out (Ccm3-iECKO). We found that Ccm3-deficient brain endothelial cells had a higher expression of genes related to the coagulation cascade and hypoxia when compared with wild-type brain endothelial cells. Immunofluorescent assays identified key molecular signatures of thrombi such as fibrin, von Willebrand factor, and activated platelets in Ccm3-iECKO mice and human CCM biopsies. Notably, we identified polyhedrocytes in Ccm3-iECKO mice and human CCM biopsies and report it for the first time. We also found that the parenchyma surrounding CCM lesions is hypoxic and that more thrombi correlate with higher levels of hypoxia. We created an in vitro model to study CCM pathology and found that human brain endothelial cells deficient for CCM3 expressed elevated levels of plasminogen activator inhibitor-1 and had a redistribution of von Willebrand factor. With transcriptomics, comprehensive imaging, and an in vitro CCM preclinical model, this study provides experimental evidence that genes and proteins related to the coagulation cascade affect the brain vasculature and promote neurological side effects such as hypoxia in CCMs. This study supports the concept that antithrombotic therapy may be beneficial for patients with CCM.

Inflammation and neutrophil extracellular traps in cerebral cavernous malformation.

Cerebral Cavernous Malformation (CCM) is a brain vascular disease with various neurological symptoms. In this study, we describe the inflammatory profile in CCM and show for the first time the formation of neutrophil extracellular traps (NETs) in rodents and humans with CCM. Through RNA-seq analysis of cerebellum endothelial cells from wild-type mice and mice with an endothelial cell-specific ablation of the Ccm3 gene (Ccm3iECKO), we show that endothelial cells from Ccm3iECKO mice have an increased expression of inflammation-related genes. These genes encode proinflammatory cytokines and chemokines, as well as adhesion molecules, which promote recruitment of inflammatory and immune cells. Similarly, immunoassays showed elevated levels of these cytokines and chemokines in the cerebellum of the Ccm3iECKO mice. Consistently, both flow cytometry and immunofluorescence analysis showed infiltration of different subsets of leukocytes into the CCM lesions. Neutrophils, which are known to fight against infection through different strategies, including the formation of NETs, represented the leukocyte subset within the most pronounced increase in CCM. Here, we detected elevated levels of NETs in the blood and the deposition of NETs in the cerebral cavernomas of Ccm3iECKO mice. Degradation of NETs by DNase I treatment improved the vascular barrier. The deposition of NETs in the cavernomas  of patients with CCM confirms the clinical relevance of NETs in CCM.

ELTD1 deletion reduces vascular abnormality and improves T-cell recruitment after PD-1 blockade in glioma.

BACKGROUND: Tumor vessels in glioma are molecularly and functionally abnormal, contributing to treatment resistance. Proteins differentially expressed in glioma vessels can change vessel phenotype and be targeted for therapy. ELTD1 (Adgrl4) is an orphan member of the adhesion G-protein-coupled receptor family upregulated in glioma vessels and has been suggested as a potential therapeutic target. However, the role of ELTD1 in regulating vessel function in glioblastoma is poorly understood. METHODS: ELTD1 expression in human gliomas and its association with patient survival was determined using tissue microarrays and public databases. The role of ELTD1 in regulating tumor vessel phenotype was analyzed using orthotopic glioma models and ELTD1-/- mice. Endothelial cells isolated from murine gliomas were transcriptionally profiled to determine differentially expressed genes and pathways. The consequence of ELTD1 deletion on glioma immunity was determined by treating tumor-bearing mice with PD-1-blocking antibodies. RESULTS: ELTD1 levels were upregulated in human glioma vessels, increased with tumor malignancy, and were associated with poor patient survival. Progression of orthotopic gliomas was not affected by ELTD1 deletion, however, tumor vascular function was improved in ELTD1-/- mice. Bioinformatic analysis of differentially expressed genes indicated increased inflammatory response and decreased proliferation in tumor endothelium in ELTD1-/- mice. Consistent with an enhanced inflammatory response, ELTD1 deletion improved T-cell infiltration in GL261-bearing mice after PD-1 checkpoint blockade. CONCLUSION: Our data demonstrate that ELTD1 participates in inducing vascular dysfunction in glioma, and suggest that targeting of ELTD1 may normalize the vessels and improve the response to immunotherapy.

Propranolol Reduces the Development of Lesions and Rescues Barrier Function in Cerebral Cavernous Malformations: A Preclinical Study.

[Figure: see text].

The blood endothelial cell chamber - An innovative system to study immune responses in drug development.

The risk for adverse immune-mediated reactions, associated with the administration of certain immunotherapeutic agents, should be mitigated early. Infusion reactions to monoclonal antibodies and other biopharmaceuticals, known as cytokine release syndrome, can arise from the release of cytokines via the drug target cell, as well as the recruitment of immune effector cells. While several in vitro cytokine release assays have been proposed up to date, many of them lack important blood components, required for this response to occur. The blood endothelial cell chamber model is an in vitro assay, composed of freshly drawn human whole blood and cultured human primary endothelial cells. Herein, its potential to study the compatibility of immunotherapeutics with the human immune system was studied by evaluating three commercially available monoclonal antibodies and bacterial endotoxin lipopolysaccharide. We demonstrate that the anti-CD28 antibody TGN1412 displayed an adaptive cytokine release profile and a distinct IL-2 response, accompanied with increased CD3+ cell recruitment. Alemtuzumab exhibited a clear cytokine response with a mixed adaptive/innate source (IFNγ, TNFα and IL-6). Its immunosuppressive nature is observed in depleted CD3+ cells. Cetuximab, associated with low infusion reactions, showed a very low or absent stimulatory effect on proinflammatory cytokines. In contrast, bacterial endotoxin demonstrated a clear innate cytokine response, defined by TNFα, IL-6 and IL-1ß release, accompanied with a strong recruitment of CD14+CD16+ cells. Therefore, the blood endothelial cell chamber model is presented as a valuable in vitro tool to investigate therapeutic monoclonal antibodies with respect to cytokine release and vascular immune cell recruitment.

Fgfbp1 promotes blood-brain barrier development by regulating collagen IV deposition and maintaining Wnt/ß-catenin signaling.

Central nervous system (CNS) blood vessels contain a functional blood-brain barrier (BBB) that is necessary for neuronal survival and activity. Although Wnt/ß-catenin signaling is essential for BBB development, its downstream targets within the neurovasculature remain poorly understood. To identify targets of Wnt/ß-catenin signaling underlying BBB maturation, we performed a microarray analysis that identified Fgfbp1 as a novel Wnt/ß-catenin-regulated gene in mouse brain endothelial cells (mBECs). Fgfbp1 is expressed in the CNS endothelium and secreted into the vascular basement membrane during BBB formation. Endothelial genetic ablation of Fgfbp1 results in transient hypervascularization but delays BBB maturation in specific CNS regions, as evidenced by both upregulation of Plvap and increased tracer leakage across the neurovasculature due to reduced Wnt/ß-catenin activity. In addition, collagen IV deposition in the vascular basement membrane is reduced in mutant mice, leading to defective endothelial cell-pericyte interactions. Fgfbp1 is required cell-autonomously in mBECs to concentrate Wnt ligands near cell junctions and promote maturation of their barrier properties in vitro Thus, Fgfbp1 is a crucial extracellular matrix protein during BBB maturation that regulates cell-cell interactions and Wnt/ß-catenin activity.

Endothelial cell clonal expansion in the development of cerebral cavernous malformations.

Cerebral cavernous malformation (CCM) is a neurovascular familial or sporadic disease that is characterised by capillary-venous cavernomas, and is due to loss-of-function mutations to any one of three CCM genes. Familial CCM follows a two-hit mechanism similar to that of tumour suppressor genes, while in sporadic cavernomas only a small fraction of endothelial cells shows mutated CCM genes. We reported that in mouse models and in human patients, endothelial cells lining the lesions have different features from the surrounding endothelium, as they express mesenchymal/stem-cell markers. Here we show that cavernomas originate from clonal expansion of few Ccm3-null endothelial cells that express mesenchymal/stem-cell markers. These cells then attract surrounding wild-type endothelial cells, inducing them to express mesenchymal/stem-cell markers and to contribute to cavernoma growth. These characteristics of Ccm3-null cells are reminiscent of the tumour-initiating cells that are responsible for tumour growth. Our data support the concept that CCM has benign tumour characteristics.

Perfusion of Porcine Kidneys With Macromolecular Heparin Reduces Early Ischemia Reperfusion Injury.

BACKGROUND: Previously, we have been able to demonstrate the possibility of coating the inner surface of the renal arteries in porcine kidneys with a heparin conjugate during hypothermic machine perfusion (HMP). The purpose of this study was to assess the efficacy of this treatment in reducing early ischemia-reperfusion injury. METHOD: Brain death was induced in male landrace pigs by stepwise volume expansion of an epidural balloon catheter until negative cerebral perfusion pressure (CPP) was obtained. Both kidneys (matched pairs; n = 6 + 6) were preserved for 20 hours by HMP during which 50 mg heparin conjugate was added to one of the HMP systems (treated group). A customized ex vivo normothermic oxygenated perfusion (NP) system with added exogenous creatinine was used to evaluate early kidney function. Blood, urine and histological samples were collected during the subsequent 3 hours of NP. RESULTS: Kidney weight was lower at the end of NP (P = 0.017) in the treated group compared with control kidneys. The rate of decline in creatinine level was faster (P = 0.024), total urinary volume was higher (P = 0.031), and the level of urine neutrophil gelatinase-associated lipocalin (NGAL) was lower (P = 0.031) in the treated group. Histologically, less tubular changes were seen (P = 0.046). During NP intrarenal resistance remained lower (P < 0.0001) in the treated group. CONCLUSIONS: Perfusion of porcine kidneys with heparin conjugate during HMP reduces preservation injury and improves organ function shortly after reperfusion. No increased risk of bleeding was seen in this setup. This protective strategy may potentially improve the quality of transplanted kidneys in the clinical setting.

Enhanced protection of the renal vascular endothelium improves early outcome in kidney transplantation: Preclinical investigations in pig and mouse.

Ischemia reperfusion injury is one of the major complications responsible for delayed graft function in kidney transplantation. Applications to reduce reperfusion injury are essential due to the widespread use of kidneys from deceased organ donors where the risk for delayed graft function is especially prominent. We have recently shown that coating of inflamed or damaged endothelial cells with a unique heparin conjugate reduces thrombosis and leukocyte recruitment. In this study we evaluated the binding capacity of the heparin conjugate to cultured human endothelial cells, to kidneys from brain-dead porcine donors, and to murine kidneys during static cold storage. The heparin conjugate was able to stably bind cultured endothelial cells with high avidity, and to the renal vasculature of explanted kidneys from pigs and mice. Treatment of murine kidneys prior to transplantation reduced platelet deposition and leukocyte infiltration 24 hours post-transplantation, and significantly improved graft function. The present study thus shows the benefits of enhanced protection of the renal vasculature during cold storage, whereby increasing the antithrombotic and anti-adhesive properties of the vascular endothelium yields improved renal function early after transplantation.

Deregulated TGF-ß/BMP Signaling in Vascular Malformations.

Correct organization of the vascular tree requires the balanced activities of several signaling pathways that regulate tubulogenesis and vascular branching, elongation, and pruning. When this balance is lost, the vessels can be malformed and fragile, and they can lose arteriovenous differentiation. In this review, we concentrate on the transforming growth factor (TGF)-ß/bone morphogenetic protein (BMP) pathway, which is one of the most important and complex signaling systems in vascular development. Inactivation of these pathways can lead to altered vascular organization in the embryo. In addition, many vascular malformations are related to deregulation of TGF-ß/BMP signaling. Here, we focus on two of the most studied vascular malformations that are induced by deregulation of TGF-ß/BMP signaling: hereditary hemorrhagic telangiectasia (HHT) and cerebral cavernous malformation (CCM). The first of these is related to loss-of-function mutation of the TGF-ß/BMP receptor complex and the second to increased signaling sensitivity to TGF-ß/BMP. In this review, we discuss the potential therapeutic targets against these vascular malformations identified so far, as well as their basis in general mechanisms of vascular development and stability.

Surface modification of pig endothelial cells with a branched heparin conjugate improves their compatibility with human blood.

Corline Heparin Conjugate (CHC), a compound of multiple unfractionated heparin chains, coats cells with a glycocalyx-like layer and may inhibit (xeno)transplant-associated activation of the plasma cascade systems. Here, we investigated the use of CHC to protect WT and genetically modified (GTKO.hCD46.hTBM) pig aortic endothelial cells (PAEC) in two pig-to-human in vitro xenotransplantation settings. Model 1: incubation of untreated or hTNFα-treated PAEC with 10% human plasma induced complement C3b/c and C5b-9 deposition, cellular activation and coagulation activation in WT and GTKO.hCD46.hTBM PAEC. Coating of untreated or hTNFα-treated PAEC with CHC (100 µg/ml) protected against human plasma-induced endothelial activation and damage. Model 2: PAEC were grown on microcarrier beads, coated with CHC, and incubated with non-anticoagulated whole human blood. Genetically modified PAEC significantly prolonged clotting time of human blood (115.0 ± 16.1 min, p < 0.001) compared to WT PAEC (34.0 ± 8.2 min). Surface CHC significantly improved the human blood compatibility of PAEC, as shown by increased clotting time (WT: 84.3 ± 11.3 min, p < 0.001; GTKO.hCD46.hTBM: 146.2 ± 20.4 min, p < 0.05) and reduced platelet adhesion, complement activation, coagulation activation and inhibition of fibrinolysis. The combination of CHC coating and genetic modification provided the greatest compatibility with human blood, suggesting that pre-transplant perfusion of genetically modified porcine organs with CHC may benefit post-transplant xenograft function.

Dangerous liaisons: complement, coagulation, and kallikrein/kinin cross-talk act as a linchpin in the events leading to thromboinflammation.

Innate immunity is fundamental to our defense against microorganisms. Physiologically, the intravascular innate immune system acts as a purging system that identifies and removes foreign substances leading to thromboinflammatory responses, tissue remodeling, and repair. It is also a key contributor to the adverse effects observed in many diseases and therapies involving biomaterials and therapeutic cells/organs. The intravascular innate immune system consists of the cascade systems of the blood (the complement, contact, coagulation, and fibrinolytic systems), the blood cells (polymorphonuclear cells, monocytes, platelets), and the endothelial cell lining of the vessels. Activation of the intravascular innate immune system in vivo leads to thromboinflammation that can be activated by several of the system's pathways and that initiates repair after tissue damage and leads to adverse reactions in several disorders and treatment modalities. In this review, we summarize the current knowledge in the field and discuss the obstacles that exist in order to study the cross-talk between the components of the intravascular innate immune system. These include the use of purified in vitro systems, animal models and various types of anticoagulants. In order to avoid some of these obstacles we have developed specialized human whole blood models that allow investigation of the cross-talk between the various cascade systems and the blood cells. We in particular stress that platelets are involved in these interactions and that the lectin pathway of the complement system is an emerging part of innate immunity that interacts with the contact/coagulation system. Understanding the resulting thromboinflammation will allow development of new therapeutic modalities.

Pleiotrophin promotes vascular abnormalization in gliomas and correlates with poor survival in patients with astrocytomas.

Glioblastomas are aggressive astrocytomas characterized by endothelial cell proliferation and abnormal vasculature, which can cause brain edema and increase patient morbidity. We identified the heparin-binding cytokine pleiotrophin as a driver of vascular abnormalization in glioma. Pleiotrophin abundance was greater in high-grade human astrocytomas and correlated with poor survival. Anaplastic lymphoma kinase (ALK), which is a receptor that is activated by pleiotrophin, was present in mural cells associated with abnormal vessels. Orthotopically implanted gliomas formed from GL261 cells that were engineered to produce pleiotrophin showed increased microvessel density and enhanced tumor growth compared with gliomas formed from control GL261 cells. The survival of mice with pleiotrophin-producing gliomas was shorter than that of mice with gliomas that did not produce pleiotrophin. Vessels in pleiotrophin-producing gliomas were poorly perfused and abnormal, a phenotype that was associated with increased deposition of vascular endothelial growth factor (VEGF) in direct proximity to the vasculature. The growth of pleiotrophin-producing GL261 gliomas was inhibited by treatment with the ALK inhibitor crizotinib, the ALK inhibitor ceritinib, or the VEGF receptor inhibitor cediranib, whereas control GL261 tumors did not respond to either inhibitor. Our findings link pleiotrophin abundance in gliomas with survival in humans and mice, and show that pleiotrophin promotes glioma progression through increased VEGF deposition and vascular abnormalization.

Mesenchymal stromal cells support endothelial cell interactions in an intramuscular islet transplantation model.

BACKGROUND: Mesenchymal stromal cells (MSC) have been under investigation for a number of therapies and have lately been in focus as immunosuppressive actors in the field of transplantation. Herein we have extended our previously published in vitro model of MSC-islets in an experimental setting of islet transplantation to the abdominal muscle. Human islets coated with luciferase-GFP transduced human MSC were transplanted to the abdomen muscle tissue of NOD-scid ILR2γ(null) mice and cellular interactions were investigated by confocal microscopy. RESULTS: The MSC reduced fibrotic encapsulation and facilitated endothelial cell interactions. In particular, we show a decreased fraction of αSMA expressing fibrotic tissue surrounding the graft in presence of MSC-islets compared to islets solely distributed into the muscle tissue. Also, in the presence of MSC, human islet endothelial cells migrated from the center of the graft out into the surrounding tissue forming chimeric blood vessels with recipient endothelial cells. Further, in the graft periphery, MSC were seen interacting with infiltrating macrophages. CONCLUSIONS: Here, in our experimental in vivo model of composite human islets and luciferase-GFP-transduced human MSC, we enable the visualization of close interactions between the MSC and the surrounding tissue. In this model of transplantation the MSC contribute to reduced fibrosis and increased islet endothelial cell migration. Furthermore, the MSC interact with the recipient vasculature and infiltrating macrophages.