Resolution of Cerebral Inflammation Following Subarachnoid Hemorrhage.

BACKGROUND: Aneurismal subarachnoid hemorrhage (SAH) is a type of hemorrhagic stroke that, despite improvement through therapeutic interventions, remains a devastating cerebrovascular disorder that has a high mortality rate and causes long-term disability. Cerebral inflammation after SAH is promoted through microglial accumulation and phagocytosis. Furthermore, proinflammatory cytokine release and neuronal cell death play key roles in the development of brain injury. The termination of these inflammation processes and restoration of tissue homeostasis are of utmost importance regarding the possible chronicity of cerebral inflammation and the improvement of the clinical outcome for affected patients post SAH. Thus, we evaluated the inflammatory resolution phase post SAH and considered indications for potential tertiary brain damage in cases of incomplete resolution. METHODS: Subarachnoid hemorrhage was induced through endovascular filament perforation in mice. Animals were killed 1, 7 and 14 days and 1, 2 and 3 months after SAH. Brain cryosections were immunolabeled for ionized calcium-binding adaptor molecule-1 to detect microglia/macrophages. Neuronal nuclei and terminal deoxyuridine triphosphate-nick end labeling staining was used to visualize secondary cell death of neurons. The gene expression of various proinflammatory mediators in brain samples was analyzed by quantitative polymerase chain reaction. RESULTS: We observed restored tissue homeostasis due to decreased microglial/macrophage accumulation and neuronal cell death 1 month after insult. However, the messenger RNA expression levels of  interleukin 6  and  tumor necrosis factor α were still elevated at 1 and 2 months post SAH, respectively. The gene expression of interleukin 1ß reached its maximum on day 1, whereas at later time points, no significant differences between the groups were detected. CONCLUSIONS: By the herein presented molecular and histological data we provide an important indication for an incomplete resolution of inflammation within the brain parenchyma after SAH. Inflammatory resolution and the return to tissue homeostasis represent an important contribution to the disease's pathology influencing the impact on brain damage and outcome after SAH. Therefore, we consider a novel complementary or even superior therapeutic approach that should be carefully rethought in the management of cerebral inflammation after SAH. An acceleration of the resolution phase at the cellular and molecular levels could be a potential aim in this context.

Minocycline Attenuates Microglia/Macrophage Phagocytic Activity and Inhibits SAH-Induced Neuronal Cell Death and Inflammation.

BACKGROUND: Neuroprotective treatment strategies aiming at interfering with either inflammation or cell death indicate the importance of these mechanisms in the development of brain injury after subarachnoid hemorrhage (SAH). This study was undertaken to evaluate the influence of minocycline on microglia/macrophage cell activity and its neuroprotective and anti-inflammatory impact 14 days after aneurismal SAH in mice. METHODS: Endovascular filament perforation was used to induce SAH in mice. SAH + vehicle-operated mice were used as controls for SAH vehicle-treated mice and SAH + minocycline-treated mice. The drug administration started 4 h after SAH induction and was daily repeated until day 7 post SAH and continued until day 14 every second day. Brain cryosections were immunolabeled for Iba1 to detect microglia/macrophages and NeuN to visualize neurons. Phagocytosis assay was performed to determine the microglia/macrophage activity status. Apoptotic cells were stained using terminal deoxyuridine triphosphate nick end labeling. Real-time quantitative polymerase chain reaction was used to estimate cytokine gene expression. RESULTS: We observed a significantly reduced phagocytic activity of microglia/macrophages accompanied by a lowered spatial interaction with neurons and reduced neuronal apoptosis achieved by minocycline administration after SAH. Moreover, the SAH-induced overexpression of pro-inflammatory cytokines and neuronal cell death was markedly attenuated by the compound. CONCLUSIONS: Minocycline treatment may be implicated as a therapeutic approach with long-term benefits in the management of secondary brain injury after SAH in a clinically relevant time window.

Physiological variables in association with spreading depolarizations in the late phase of ischemic stroke.

Physiological effects of spreading depolarizations (SD) are only well studied in the first hours after experimental stroke. In patients with malignant hemispheric stroke (MHS), monitoring of SDs is restricted to the postoperative ICU stay, typically day 2-7 post-ictus. Therefore, we investigated the role of physiological variables (temperature, intracranial pressure, mean arterial pressure and cerebral perfusion pressure) in relationship to SD during the late phase after MHS in humans. Additionally, an experimental stroke model was used to investigate hemodynamic consequences of SD during this time window. In 60 patients with MHS, the occurrence of 1692 SDs was preceded by a decrease in mean arterial pressure (-1.04 mmHg; p = .02) and cerebral perfusion pressure (-1.04 mmHg; p = .03). Twenty-four hours after middle cerebral artery occlusion in 50 C57Bl6/J mice, hypothermia led to prolonged SD-induced hyperperfusion (+2.8 min; p < .05) whereas hypertension mitigated initial hypoperfusion (-1.4 min and +18.5%Δ rCBF; p < .01). MRI revealed that SDs elicited 24 hours after experimental stroke were associated with lesion progression (15.9 vs. 14.8 mm³; p < .01). These findings of small but significant effects of physiological variables on SDs in the late phase after ischemia support the hypothesis that the impact of SDs may be modified by adjusting physiological variables.

Three-Dimensional Iron Oxide Nanoparticle-Based Contrast-Enhanced Magnetic Resonance Imaging for Characterization of Cerebral Arteriogenesis in the Mouse Neocortex.

Purpose: Subsurface blood vessels in the cerebral cortex have been identified as a bottleneck in cerebral perfusion with the potential for collateral remodeling. However, valid techniques for non-invasive, longitudinal characterization of neocortical microvessels are still lacking. In this study, we validated contrast-enhanced magnetic resonance imaging (CE-MRI) for in vivo characterization of vascular changes in a model of spontaneous collateral outgrowth following chronic cerebral hypoperfusion. Methods: C57BL/6J mice were randomly assigned to unilateral internal carotid artery occlusion or sham surgery and after 21 days, CE-MRI based on T2*-weighted imaging was performed using ultra-small superparamagnetic iron oxide nanoparticles to obtain subtraction angiographies and steady-state cerebral blood volume (ss-CBV) maps. First pass dynamic susceptibility contrast MRI (DSC-MRI) was performed for internal validation of ss-CBV. Further validation at the histological level was provided by ex vivo serial two-photon tomography (STP). Results: Qualitatively, an increase in vessel density was observed on CE-MRI subtraction angiographies following occlusion; however, a quantitative vessel tracing analysis was prone to errors in our model. Measurements of ss-CBV reliably identified an increase in cortical vasculature, validated by DSC-MRI and STP. Conclusion: Iron oxide nanoparticle-based ss-CBV serves as a robust, non-invasive imaging surrogate marker for neocortical vessels, with the potential to reduce and refine preclinical models targeting the development and outgrowth of cerebral collateralization.

Tumor-Associated Microglia/Macrophages as a Predictor for Survival in Glioblastoma and Temozolomide-Induced Changes in CXCR2 Signaling with New Resistance Overcoming Strategy by Combination Therapy.

Tumor recurrence is the main challenge in glioblastoma (GBM) treatment. Gold standard therapy temozolomide (TMZ) is known to induce upregulation of IL8/CXCL2/CXCR2 signaling that promotes tumor progression and angiogenesis. Our aim was to verify the alterations on this signaling pathway in human GBM recurrence and to investigate the impact of TMZ in particular. Furthermore, a combi-therapy of TMZ and CXCR2 antagonization was established to assess the efficacy and tolerability. First, we analyzed 76 matched primary and recurrent GBM samples with regard to various histological aspects with a focus on the role of TMZ treatment and the assessment of predictors of overall survival (OS). Second, the combi-therapy with TMZ and CXCR2-antagonization was evaluated in a syngeneic mouse tumor model with in-depth immunohistological investigations and subsequent gene expression analyses. We observed a significantly decreased infiltration of tumor-associated microglia/macrophages (TAM) in recurrent tumors, while a high TAM infiltration in primary tumors was associated with a reduced OS. Additionally, more patients expressed IL8 in recurrent tumors and TMZ therapy maintained CXCL2 expression. In mice, enhanced anti-tumoral effects were observed after combi-therapy. In conclusion, high TAM infiltration predicts a survival disadvantage, supporting findings of the tumor-promoting phenotype of TAMs. Furthermore, the combination therapy seemed to be promising to overcome CXCR2-mediated resistance.

Low-dose rapamycin does not impair vascular integrity and tubular regeneration after kidney transplantation in rats.

mTOR inhibitors offer advantages after kidney transplantation including antiviral and antitumor activity besides facilitating low calcineurin inhibitor exposure to reduce nephrotoxicity. Concerns about adverse effects due to antiproliferative and antiangiogenic properties have limited their clinical use particularly early after transplantation. Interference with vascular endothelial growth factor (VEGF)-A, important for physiologic functioning of renal endothelial cells and tubular epithelium, has been implicated in detrimental renal effects of mTOR inhibitors. Low doses of Rapamycin (loading dose 3 mg/kg bodyweight, daily doses 1.5 mg/kg bodyweight) were administered in an allogenic rat kidney transplantation model resulting in a mean through concentration of 4.30 ng/mL. Glomerular and peritubular capillaries, tubular cell proliferation, or functional recovery from preservation/reperfusion injury were not compromised in comparison to vehicle treated animals. VEGF-A, VEGF receptor 2, and the co-receptor Neuropilin-1 were upregulated by Rapamycin within 7 days. Rat proximal tubular cells (RPTC) responded in vitro to hypoxia with increased VEGF-A and VEGF-R1 expression that was not suppressed by Rapamycin at therapeutic concentrations. Rapamycin did not impair proliferation of RPTC under hypoxic conditions. Low-dose Rapamycin early posttransplant does not negatively influence the VEGF network crucial for recovery from preservation/reperfusion injury. Enhancement of VEGF signaling peritransplant holds potential to further improve outcomes.

Establishment and Validation of CyberKnife Irradiation in a Syngeneic Glioblastoma Mouse Model.

CyberKnife stereotactic radiosurgery (CK-SRS) precisely delivers radiation to intracranial tumors. However, the underlying radiobiological mechanisms at high single doses are not yet fully understood. Here, we established and evaluated the early radiobiological effects of CK-SRS treatment at a single dose of 20 Gy after 15 days of tumor growth in a syngeneic glioblastoma-mouse model. Exact positioning was ensured using a custom-made, non-invasive, and trackable frame. One superimposed target volume for the CK-SRS planning was created from the fused tumor volumes obtained from MRIs prior to irradiation. Dose calculation and delivery were planned using a single-reference CT scan. Six days after irradiation, tumor volumes were measured using MRI scans, and radiobiological effects were assessed using immunofluorescence staining. We found that CK-SRS treatment reduced tumor volume by approximately 75%, impaired cell proliferation, diminished tumor vasculature, and increased immune response. The accuracy of the delivered dose was demonstrated by staining of DNA double-strand breaks in accordance with the planned dose distribution. Overall, we confirmed that our proposed setup enables the precise irradiation of intracranial tumors in mice using only one reference CT and superimposed MRI volumes. Thus, our proposed mouse model for reproducible CK-SRS can be used to investigate radiobiological effects and develop novel therapeutic approaches.

Endovascular Perforation Model for Subarachnoid Hemorrhage Combined with Magnetic Resonance Imaging (MRI).

The endovascular filament perforation model to mimic subarachnoid hemorrhage (SAH) is a commonly used model - however, the technique can cause a high mortality rate as well as an uncontrollable volume of SAH and other intracranial complications such as stroke or intracranial hemorrhage. In this protocol, a standardized SAH mouse model is presented, induced by endovascular filament perforation, combined with magnetic resonance imaging (MRI) 24 h after operation to ensure the correct bleeding site and exclude other relevant intracranial pathologies. Briefly, C57BL/6J mice are anesthetized with an intraperitoneal ketamine/xylazine (70 mg/16 mg/kg body weight) injection and placed in a supine position. After midline neck incision, the common carotid artery (CCA) and carotid bifurcation are exposed, and a 5-0 non-absorbable monofilament polypropylene suture is inserted in a retrograde fashion into the external carotid artery (ECA) and advanced into the common carotid artery. Then, the filament is invaginated into the internal carotid artery (ICA) and pushed forward to perforate the anterior cerebral artery (ACA). After recovery from surgery, mice undergo a 7.0 T MRI 24 h later. The volume of bleeding can be quantified and graded via postoperative MRI, enabling a robust experimental SAH group with the option to perform further subgroup analyses based on blood quantity.

The CXCR2/CXCL2 signalling pathway - An alternative therapeutic approach in high-grade glioma.

OBJECTIVE: Besides VEGF, alternative signalling via CXCR2 and its ligands CXCL2/CXCL8 is a crucial part of angiogenesis in glioblastoma. Our aim was to understand the role of CXCR2 for glioma biology and elucidate the therapeutic potential of its specific inhibition. METHODS: GL261 glioma cells were implanted intracranially in syngeneic mice. The 14 or 7 days of local or systemic treatment with CXCR2-antagonist (SB225002) was initiated early on the day of tumour cell implantation or delayed after 14 days of tumour growth. Glioma volume was verified using MRI before and after treatment. Immunofluorescence staining was used to investigate tumour progression, angiogenesis and microglial behaviour. Furthermore, in vitro assays and gene expression analyses of glioma and endothelial cells were performed to validate inhibitor activity. RESULTS: CXCR2-blocking led to significantly reduced glioma volumes of around 50% after early and delayed local treatments. The treated tumours were comparable with controls regarding invasiveness, proliferation and apoptotic cell activity. Furthermore, no differences in CXCR2/CXCL2 expression were observed. However, immunostaining revealed reduction in vessel density and accumulation of microglia/macrophages, whereas interaction of these myeloid cells with tumour vessels was enhanced. In vitro analyses of the CXCR2-antagonist showed its direct impact on proliferation of glioma and endothelial cells if used at higher concentrations. In addition, expression of CXCR2/CXCL2 signalling genes was increased in both cell types by SB225002, but VEGF-relevant genes were unaffected. CONCLUSION: The CXCR2-antagonist inhibited glioma growth during tumour initiation and progression, whereas treatment was well-tolerated by the recipients. Thus, the CXCR2/CXCL2 signalling represents a promising therapeutic target in glioma.

Pigment Epithelium-Derived Factor Improves Paracellular Blood-Brain Barrier Integrity in the Normal and Ischemic Mouse Brain.

Pigment epithelium-derived factor (PEDF) is a neurotrophic factor with neuroprotective, antiangiogenic, and antipermeability effects. In the brain, blood-brain barrier (BBB) function is essential for homeostasis. Its impairment plays a crucial role in the pathophysiology of many neurological diseases, including ischemic stroke. We investigated (a) whether PEDF counteracted vascular endothelial growth factor (VEGF)-induced BBB disruption in the mouse brain, (b) the time course and route of BBB permeability and the dynamics of PEDF expression after cerebral ischemia, and (c) whether intraventricular infusion of PEDF ameliorated brain ischemia by reducing BBB impairment. C57Bl6/N mice received intraparenchymal injections of CSF, VEGF, or a combination of VEGF and PEDF. PEDF increased paracellular but not transcellular BBB integrity as indicated by an increase in the tight junction protein claudin-5. In another group of mice undergoing 60-min middle cerebral artery occlusion (MCAO), transcellular BBB permeability (fibrinogen staining in the absence of a loss of claudin-5) increased as early as 6 h after reperfusion. PEDF immunofluorescence increased at 24 h, which paralleled with a decreased paracellular BBB permeability (claudin-5). PEDF after MCAO originated from the blood stream and endogenous pericytes. In the third experiment, the intraventricular infusion of PEDF decreased edema and cell death after MCAO, potentially mediated by the improvement of the paracellular route of BBB permeability (claudin-5) in the absence of an amelioration of Evans Blue extravasation. Together, our data suggest that PEDF improves BBB function after cerebral ischemia by affecting the paracellular but not the transcellular route. However, further quantitative data of the different routes of BBB permeability will be required to validate our findings.

Loss of the transcription factor RBPJ induces disease-promoting properties in brain pericytes.

Sufficient vascular supply is indispensable for brain development and function, whereas dysfunctional blood vessels are associated with human diseases such as vascular malformations, stroke or neurodegeneration. Pericytes are capillary-associated mesenchymal cells that limit vascular permeability and protect the brain by preserving blood-brain barrier integrity. Loss of pericytes has been linked to neurodegenerative changes in genetically modified mice. Here, we report that postnatal inactivation of the Rbpj gene, encoding the transcription factor RBPJ, leads to alteration of cell identity markers in brain pericytes, increases local TGFß signalling, and triggers profound changes in endothelial behaviour. These changes, which are not mimicked by pericyte ablation, imperil vascular stability and induce the acquisition of pathological landmarks associated with cerebral cavernous malformations. In adult mice, loss of Rbpj results in bigger stroke lesions upon ischemic insult. We propose that brain pericytes can acquire deleterious properties that actively enhance vascular lesion formation and promote pathogenic processes.

In vitro and in vivo testing of a novel local nicardipine delivery system to the brain: a preclinical study.

OBJECTIVE: The management of patients with aneurysmal subarachnoid hemorrhage (aSAH) remains a highly demanding challenge in critical care medicine. Despite all efforts, the calcium channel antagonist nimodipine remains the only drug approved for improving outcomes after aSAH. However, in its current form of application, it provides less than optimal efficacy and causes dose-limiting hypotension in a substantial number of patients. Here, the authors tested in vitro the release dynamics of a novel formulation of the calcium channel blocker nicardipine and in vivo local tolerance and tissue reaction using a chronic cranial window model in mice. METHODS: To characterize the release kinetics in vitro, dissolution experiments were performed using artificial cerebrospinal fluid over a time period of 21 days. The excipients used in this formulation (NicaPlant) for sustained nicardipine release are a mixture of two completely degradable polymers. A chronic cranial window in C57BL/6 mice was prepared, and NicaPlant slices were placed in proximity to the exposed cerebral vasculature. Epifluorescence video microscopy was performed right after implantation and on days 3 and 7 after surgery. Vessel diameter of the arteries and veins, vessel permeability, vessel configuration, and leukocyte-endothelial cell interaction were quantified by computer-assisted analysis. Immunofluorescence staining was performed to analyze inflammatory reactions and neuronal alterations. RESULTS: In vitro the nicardipine release profile showed an almost linear curve with about 80% release at day 15 and full release at day 21. In vivo epifluorescence video microscopy showed a significantly higher arterial vessel diameter in the NicaPlant group due to vessel dilatation (21.6 ± 2.6 µm vs 17.8 ± 1.5 µm in controls, p < 0.01) confirming vasoactivity of the implant, whereas the venous diameter was not affected. Vessel dilatation did not have any influence on the vessel permeability measured by contrast extravasation of the fluorescent dye in epifluorescence microscopy. Further, an increased leukocyte-endothelial cell interaction due to the implant could not be detected. Histological analysis did not show any microglial activation or accumulation. No structural neuronal changes were observed. CONCLUSIONS: NicaPlant provides continuous in vitro release of nicardipine over a 3-week observation period. In vivo testing confirmed vasoactivity and lack of toxicity. The local application of this novel nicardipine delivery system to the subarachnoid space is a promising tool to improve patient outcomes while avoiding systemic side effects.

Targeting the extradomain A of fibronectin allows identification of vascular resistance to antiangiogenic therapy in experimental glioma.

INTRODUCTION: Clinical application of antiangiogenic therapy lacks direct visualization of therapy efficacy and vascular resistance. We aimed to establish molecular imaging during treatment with sunitinib using the fibronectin extradomain A specific small immunoprotein(SIP)-F8 in glioma. METHODS: Biodistribution analysis of F8-SIP-Alexa-555 was performed in SF126-glioma bearing or control mice (n = 23 and 7, respectively). Intravital microscopy(IVM) was performed on a microvascular level after 7 days (n = 5 per group) and subsequently after 6 days of sunitinib treatment (n = 4) or without (n = 2).Additionally, near infrared fluorescence(NIRF) imaging was established with F8-SIP-Alexa-750 allowing non-invasive imaging with and without antiangiogenic treatment in orthotopic tumors (n = 38 divided in 4 groups). MRI was used to determine tumor size and served as a reference for NIRF imaging. RESULTS: F8-SIP demonstrated a time and hemodynamic dependent tumor specific accumulation. A significantly higher vascular accumulation occurred with antiangiogenic treatment compared to untreated tumors enabling visualization of resistant tumor vessels by F8-SIP-mediated NIRF imaging. In orthotopic tumors, sunitinib reduced F8-SIP-Alexa-750 enrichment volume but not fluorescence intensity indicative of F8-SIP accumulation in fewer vessels. CONCLUSION: F8-SIP is highly tumor specific with time and hemodynamic dependent biodistribution. The higher vascular accumulation to remaining vessels enables molecular imaging and targeting of therapy resistant tumor vessels.

Interleukin 6-Mediated Endothelial Barrier Disturbances Can Be Attenuated by Blockade of the IL6 Receptor Expressed in Brain Microvascular Endothelial Cells.

Compromised blood-brain barrier (BBB) by dysregulation of cellular junctions is a hallmark of many cerebrovascular disorders due to the pro-inflammatory cytokines action. Interleukin 6 (IL6) is implicated in inflammatory processes and in secondary brain injury after subarachnoid hemorrhage (SAH) but its role in the maintenance of cerebral endothelium still requires a precise elucidation. Although IL6 has been shown to exert pro-inflammatory action on brain microvascular endothelial cells (ECs), the expression of one of the IL6 receptors, the IL6R is controversially discussed. In attempt to reach more clarity in this issue, we present here an evident baseline expression of the IL6R in BBB endothelium in vivo and in an in vitro model of the BBB, the cEND cell line. A significantly increased expression of IL6R and its ligand was observed in BBB capillaries 2 days after experimental SAH in mice. In vitro, we saw IL6 administration resulting in an intracellular and extracellular elevation of IL6 protein, which was accompanied by a reduced expression of tight and adherens junctions, claudin-5, occludin, and vascular-endothelial (VE-) cadherin. By functional assays, we could demonstrate IL6-incubated brain ECs to lose their endothelial integrity that can be attenuated by inhibiting the IL6R. Blockade of the IL6R by a neutralizing antibody has reconstituted the intercellular junction expression to the control level and caused a restoration of the transendothelial electrical resistance of the cEND cell monolayer. Our findings add depth to the current understanding of the involvement of the endothelial IL6R in the loss of EC integrity implicating potential therapy options.

EphrinB2 Activation Enhances Vascular Repair Mechanisms and Reduces Brain Swelling After Mild Cerebral Ischemia.

OBJECTIVE: Cerebral edema caused by the disruption of the blood-brain barrier is a major complication after stroke. Therefore, strategies to accelerate and enhance neurovascular recovery after stroke are of prime interest. Our main aim was to study the role of ephrinB2/EphB4 signaling in mediating the vascular repair and in blood-brain barrier restoration after mild cerebral ischemia occlusion/reperfusion. APPROACH AND RESULTS: Here, we show that the guidance molecule ephrinB2 plays a key role in neurovascular protection and blood-brain barrier restoration after stroke. In a focal stroke model, we characterize the stroke-induced damage to cerebral blood vessels and their subsequent endogenous repair on a cellular, molecular, and functional level. EphrinB2 and its tyrosine kinase receptor EphB4 are upregulated early after stroke by endothelial cells and perivascular support cells, in parallel to their reassembly during neurovascular recovery. Using both retroviral and pharmacological approaches, we show that the inhibition of ephrinB2/EphB4 signaling suppresses post-middle cerebral artery occlusion neurovascular repair mechanisms resulting in an aggravation of brain swelling. In contrast, the activation of ephrinB2 after brain ischemia leads to an increased pericyte recruitment and increased endothelial-pericyte interaction, resulting in an accelerated neurovascular repair after ischemia. CONCLUSIONS: We show that reducing swelling could result in improved outcome because of reduction in damaged brain tissue. We also identify a novel role for ephrinB2/EphB4 signaling in the maintenance of the neurovascular homeostasis and provide a novel therapeutic approach in reducing brain swelling after stroke.

Esm1 modulates endothelial tip cell behavior and vascular permeability by enhancing VEGF bioavailability.

RATIONALE: Endothelial cell-specific molecule 1 (Esm1) is a secreted protein thought to play a role in angiogenesis and inflammation. However, there is currently no direct in vivo evidence supporting a function of Esm1 in either of these processes. OBJECTIVE: To determine the role of Esm1 in vivo and the underlying molecular mechanisms. METHODS AND RESULTS: We generated and analyzed Esm1 knockout (Esm1(KO)) mice to study its role in angiogenesis and inflammation. Esm1 expression is induced by the vascular endothelial growth factor A (VEGF-A) in endothelial tip cells of the mouse retina. Esm1(KO) mice showed delayed vascular outgrowth and reduced filopodia extension, which are both VEGF-A-dependent processes. Impairment of Esm1 function led to a decrease in phosphorylated Erk1/2 (extracellular-signal regulated kinases 1/2) in sprouting vessels. We also found that Esm1(KO) mice displayed a 40% decrease in leukocyte transmigration. Moreover, VEGF-induced vascular permeability was decreased by 30% in Esm1(KO) mice and specifically on stimulation with VEGF-A165 but not VEGF-A121. Accordingly, cerebral edema attributable to ischemic stroke-induced vascular permeability was reduced by 50% in the absence of Esm1. Mechanistically, we show that Esm1 binds directly to fibronectin and thereby displaces fibronectin-bound VEGF-A165 leading to increased bioavailability of VEGF-A165 and subsequently enhanced levels of VEGF-A signaling. CONCLUSIONS: Esm1 is simultaneously a target and modulator of VEGF signaling in endothelial cells, playing a role in angiogenesis, inflammation, and vascular permeability, which might be of potential interest for therapeutic applications.

Renal ischemia and transplantation predispose to vascular constriction mediated by angiotensin II type 1 receptor-activating antibodies.

BACKGROUND: We previously described angiotensin II type 1 receptor-activating antibodies (AT1R-Abs) in renal transplant recipients with vascular rejection and malignant hypertension. In this study, we tested the hypothesis that AT1R-Abs can cause renal artery contraction by AT1R activation with renal ischemia representing a key permissive factor and therefore contribute to renal pathologic condition. METHODS: Isolated renal and mesenteric arteries from Lewis rats were incubated with purified AT1R-Abs from patients with human leukocyte antigen antibody-negative vascular rejection. Vascular contraction was measured using small vessel myography. The measurements were repeated with renal arteries derived from native kidneys subjected to ischemia-reperfusion or after transplantation in a low-responder Fischer 344-to-Lewis rat kidney-transplantation model. RESULTS: AT1R-Abs acted in a vascular bed-specific manner and caused small contractions only in native rat renal arteries but not in mesenteric arteries. AT1R-Abs did not alter the vascular reactivity to phenylephrine, angiotensin II, or acetylcholine in native renal arteries. In contrast, AT1R-Abs caused a pronounced (>10-fold) contraction of renal arteries after ischemia and after allogeneic transplantation. Pretreatment with pharmacologic AT1R blocker only partially inhibited the AT1R-Abs-induced contraction, which was almost completely abolished by neutralizing peptides targeting epitopes of AT1R-Abs on the second loop of AT1R. CONCLUSIONS: These data demonstrate that AT1R-Abs can induce renal vascular contraction under predisposing conditions such as in ischemic or transplanted kidneys. Neutralizing antibodies against specific epitopes in the AT1R can ameliorate this contraction.

Cytoprotective Actions of FTY720 Modulate Severe Preservation Reperfusion Injury in Rat Renal Transplants.

BACKGROUND: Fingolimod (FTY720) is a potent agonist of sphingosine 1 phosphate receptors and thereby interferes with lymphocyte trafficking. We previously showed that FTY720 protects from mild preservation reperfusion injury induced by 4 hr of cold ischemia. The purpose of this study was to explore the role of FTY720 in ischemic injury and regeneration using a clinically relevant rat renal transplant model with 24 hr of cold ischemia. METHODS: Donor kidneys were cold stored in the University of Wisconsin solution for 24 hr before transplantation into bilaterally nephrectomized syngeneic recipients (n=6 per group), which received 0.5 mg/kg/d FTY720 or vehicle through oral gavage. Grafts were harvested 2 or 7 days posttransplantation. Renal tissue was examined histologically, stained for apoptosis, proliferation, inflammatory cell infiltrates, and studied for transforming growth factor-beta, and tumor necrosis factor-alpha expression. Rat proximal tubular cells were incubated with 0.1 to 30 micromol/L of phosphorylated FTY720 to test for in vitro cytopathic effects. RESULTS: FTY720 induced peripheral lymphopenia and significantly reduced intragraft CD3 and ED1 infiltrates. Acute tubular damage scores and graft function were not influenced by FTY720. Tubular apoptosis was significantly reduced, whereas the number of proliferating cell nuclear antigen-positive tubular cells were markedly increased. FTY720 attenuated renal tumor necrosis factor-alpha and transforming growth factor-beta expression. In vitro, pharmacologic concentrations up to 1 micromol/L of phosphorylated FTY720 did not affect tubular cell viability. CONCLUSION: FTY720 confers tubular epithelial protection in the presence of severe preservation reperfusion injury. Beneficial effects may in part be due to reduction in cell-mediated immune mechanisms. Furthermore, FTY720 could be helpful in patients with delayed graft function.

Mechanisms of tubular volume retention in immune-mediated glomerulonephritis.

Glomerulonephritis is characterized by hematuria, proteinuria, hypertension, and edema, but the mechanisms contributing to volume disorders are controversial. Here we used the rat anti-Thy1 model of mesangioproliferative glomerulonephritis to test the hypothesis that disturbed salt and water homeostasis is based on tubular epithelial changes that cause salt retention. In this model there was an early onset of pronounced proteinuria and lipiduria associated with reduced fractional sodium excretion and a lowering of the renin-angiotensin-aldosterone system. The glomerular filtration rate and creatinine clearance were decreased on day 6. There was a reduced abundance of the major salt and water transport proteins on the proximal tubular brush border membrane and which paralleled cellular protein overload, enhanced membrane cholesterol uptake and cytoskeletal changes. Alterations in thick ascending limb were moderate. Changes in the collecting ducts were characterized by an enhanced abundance and increased subunit cleavage of the epithelial sodium channel, both events consistent with increased sodium reabsorption. We suggest that irrespective of the proximal tubular changes, altered collecting duct sodium reabsorption may be crucial for volume retention in acute glomerulonephritis. We suggest that enhanced proteolytic cleavage of ion transporter subunits might be a novel mechanism of channel activation in glomerular diseases. Whether these proteases are filtered or locally secreted awaits determination.

Angiotensin II type 1-receptor activating antibodies in renal-allograft rejection.

BACKGROUND: Antibodies against HLA antigens cause refractory allograft rejection with vasculopathy in some, but not all, patients. METHODS: We studied 33 kidney-transplant recipients who had refractory vascular rejection. Thirteen had donor-specific anti-HLA antibodies, whereas 20 did not. Malignant hypertension was present in 16 of the patients without anti-HLA antibodies, 4 of whom had seizures. The remaining 17 patients had no malignant hypertension. We hypothesized that activating antibodies targeting the angiotensin II type 1 (AT1) receptor might be involved. RESULTS: Activating IgG antibodies targeting the AT1 receptor were detected in serum from all 16 patients with malignant hypertension and without anti-HLA antibodies, but in no other patients. These receptor-activating antibodies are subclass IgG1 and IgG3 antibodies that bind to two different epitopes on the second extracellular loop of the AT1 receptor. Tissue factor expression was increased in renal-biopsy specimens from patients with these antibodies. In vitro stimulation of vascular cells with an AT1-receptor-activating antibody induced phosphorylation of ERK 1/2 kinase and increased the DNA binding activity of the transcription factors activator protein 1 (AP-1) and nuclear factor-kappaB. The AT1 antagonist losartan blocked agonistic AT1-receptor antibody-mediated effects, and passive antibody transfer induced vasculopathy and hypertension in a rat kidney-transplantation model. CONCLUSIONS: A non-HLA, AT1-receptor-mediated pathway may contribute to refractory vascular rejection, and affected patients might benefit from removal of AT1-receptor antibodies or from pharmacologic blockade of AT1 receptors.