Anti-osteopontin therapy leads to improved edema and infarct size in a murine model of ischemic stroke.

Ischemic stroke is a serious neurological disorder that is associated with dysregulation of the neurovascular unit (NVU) and impairment of the blood-brain barrier (BBB). Paradoxically, reperfusion therapies can aggravate NVU and BBB dysfunction, leading to deleterious consequences in addition to the obvious benefits. Using the recently established EPAM-ia method, we identified osteopontin as a target dysregulated in multiple NVU cell types and demonstrated that osteopontin targeting in the early acute phase post-transient middle cerebral artery occlusion (tMCAO) evolves protective effects. Here, we assessed the time course of osteopontin and CD44 receptor expression in NVU cells and examined cerebroprotective effects of osteopontin targeting in early and late acute phases of ischemic stroke. Expression analysis of osteopontin and CD44 receptor post-tMCAO indicated increased levels of both, from early to late acute phases, which was supported by their co-localization in NVU cells. Combined osteopontin targeting in early and late acute phases with anti-osteopontin antibody resulted in further improvement in BBB recovery and edema reduction compared to targeting only in the early acute phase comprising the reperfusion window. Combined targeting led to reduced infarct volumes, which was not observed for the single early acute phase targeting. The effects of the therapeutic antibody were confirmed both in vitro and in vivo in reducing osteopontin and CD44 expression. Osteopontin targeting at the NVU in early and late acute phases of ischemic stroke improves edema and infarct size in mice, suggesting anti-osteopontin therapy as promising adjunctive treatment to reperfusion therapy.

Profiling the neurovascular unit unveils detrimental effects of osteopontin on the blood-brain barrier in acute ischemic stroke.

Blood-brain barrier (BBB) dysfunction, characterized by degradation of BBB junctional proteins and increased permeability, is a crucial pathophysiological feature of acute ischemic stroke. Dysregulation of multiple neurovascular unit (NVU) cell types is involved in BBB breakdown in ischemic stroke that may be further aggravated by reperfusion therapy. Therefore, therapeutic co-targeting of dysregulated NVU cell types in acute ischemic stroke constitutes a promising strategy to preserve BBB function and improve clinical outcome. However, methods for simultaneous isolation of multiple NVU cell types from the same diseased central nervous system (CNS) tissue, crucial for the identification of therapeutic targets in dysregulated NVU cells, are lacking. Here, we present the EPAM-ia method, that facilitates simultaneous isolation and analysis of the major NVU cell types (endothelial cells, pericytes, astrocytes and microglia) for the identification of therapeutic targets in dysregulated NVU cells to improve the BBB function. Applying this method, we obtained a high yield of pure NVU cells from murine ischemic brain tissue, and generated a valuable NVU transcriptome database ( https://bioinformatics.mpi-bn.mpg.de/SGD_Stroke ). Dissection of the NVU transcriptome revealed Spp1, encoding for osteopontin, to be highly upregulated in all NVU cells 24 h after ischemic stroke. Upregulation of osteopontin was confirmed in stroke patients by immunostaining, which was comparable with that in mice. Therapeutic targeting by subcutaneous injection of an anti-osteopontin antibody post-ischemic stroke in mice resulted in neutralization of osteopontin expression in the NVU cell types investigated. Apart from attenuated glial activation, osteopontin neutralization was associated with BBB preservation along with decreased brain edema and reduced risk for hemorrhagic transformation, resulting in improved neurological outcome and survival. This was supported by BBB-impairing effects of osteopontin in vitro. The clinical significance of these findings is that anti-osteopontin antibody therapy might augment current approved reperfusion therapies in acute ischemic stroke by minimizing deleterious effects of ischemia-induced BBB disruption.

C5b-9 deposits on endomysial capillaries in non-dermatomyositis cases.

Deposits of the terminal-membrane-attack-complex (MAC) C5b-9 on perfascicular endomysial capillaries are generally regarded as diagnostic hallmark of dermatomyositis (DM). Although the pathophysiology is not clear, C5b-9 deposits on capillaries seem to be associated with microinfarctions and vascular damage. Here, we report on a series of 19 patients presenting with C5b-9 accumulation on endomysial capillaries in the absence of features for DM. To decipher differences in the capillary C5b-9 accumulation pattern between DM and non-DM cases, we assessed the extent of endomysial capillary C5b-9 deposits related to capillary density and extent of myofiber necrosis by immunohistochemistry in 12 DM and 8 control patients. We found similar numbers of C5b-9-positive myofibers in both DM and non-DM C5b-9(+) cases. The distribution pattern differed as DM cases showed significantly more perifascicular capillary C5b-9 deposits as compared to non-DM cases, which presented stronger endomysial capillary C5b-9 deposits in a diffuse pattern. While total capillary density was not differing, DM patients displayed significantly more C5b-9(+) necrotic fibers as compared to non-DM C5b-9(+). In summary, endomysial capillary C5b-9 deposits are present in a variety of non-DM cases, however with differing distribution pattern. In conclusion, capillary C5b-9(+) deposits should be assessed critically, taking into consideration the distribution pattern.

Differential expression of vascular endothelial growth factor A, its receptors VEGFR-1, -2, and -3 and co-receptors neuropilin-1 and -2 does not predict bevacizumab response in human astrocytomas.

BACKGROUND: A major hallmark of malignant progression in human astrocytomas is the formation of new blood vessels. Antiangiogenic therapy using the anti-vascular endothelial growth factor (VEGF)-antibody bevacizumab leads to increased progression-free survival in glioblastoma patients but does not influence their overall survival. To date, it is unclear why antiangiogenic therapy fails in many glioblastoma patients, while a small subpopulation profits considerably from this treatment. METHODS: The aim of our study was to determine the expression of VEGF-A and its (co-) receptors by immunohistochemistry and to test the association with patient survival in 350 glioma patients. Additionally, VEGF-A expression was analyzed by in-situ hybridization. In 18 patients, the protein expression was compared with the bevacizumab response according to extended and modified RANO criteria. RESULTS: We found a heterogeneous expression pattern of VEGF and its receptors in glioblastoma patients with significantly lower levels in WHO grade II and III tumors and normal-appearing brain tissue (P < .001). Pilocytic astrocytomas (WHO grade I) showed significantly higher VEGFR-1, -2 and neuropilin-1 levels as compared to WHO grade II and III astrocytomas (P < .01) but at lower levels than glioblastomas. The expression of neuropilin-2 was low in all tumors. There was neither a significant correlation between protein expression and patient survival nor between protein levels and bevacizumab response after modified RANO criteria. CONCLUSION: Since our data indicate that beneficial response to bevacizumab treatment is independent of the expression of VEGF-A and its (co-) receptors, further investigation is needed to decipher the underlying mechanisms of antiangiogenic treatment response.

Confounding factors in diagnostics of MGMT promoter methylation status in glioblastomas in stereotactic biopsies.

BACKGROUND: In nonresectable glioblastoma (GBM), stereotactic biopsies are performed to retrieve tissue for diagnostic purposes. The analysis of O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation adds prognostic and predictive information. OBJECTIVES: The aim of the study was to detect confounding factors that limit the number of conclusive MGMT promoter methylation results. METHODS: We analyzed 71 consecutive GBM patients undergoing stereotactic biopsy on whom MGMT analysis was performed by methylation-specific polymerase chain reaction. Specimens were correlated to imaging by coregistration and prospective documentation of biopsy localization. Our findings were validated in an additional 62 GBM stereotactic biopsies. RESULTS: Our results demonstrate that the best MGMT promoter methylation results were obtained from samples (n = 71) taken in a tangential manner from tumor areas showing contrast enhancement in magnetic resonance imaging. In the additional validation series of 62 stereotactically biopsied GBM, we were able to increase the rate of conclusive MGMT promoter methylation results from 76.1 to 85.48% by strictly planning the route of biopsy in a tangential manner if possible. CONCLUSIONS: These results underline that within the contrast-enhanced tumor part, choosing the trajectory in a tangential manner increases the diagnostic yield for conclusive MGMT promoter methylation analyses in stereotactic biopsies as a basis for patient stratification and individualized therapy.