Anil Zechariah

Implications of vascular endothelial growth factor for postischemic neurovascular remodeling

Neurovascular remodeling has been recently recognized as a promising target for neurologic therapies. Hopes have emerged that, by stimulating vessel growth, it may be possible to stabilize brain perfusion, and at the same time promote neuronal survival, brain plasticity, and neurologic recovery. In this review, we outline the role of vascular endothelial growth factor (VEGF) in the ischemic brain, analyzing how this growth factor contributes to brain remodeling. Studies with therapeutic VEGF administration resulted in quite variable results depending on the route and time point of delivery. Local VEGF administration consistently enhanced neurologic recovery, whereas acute intravenous delivery exacerbated brain infarcts due to enhanced brain edema. Future studies should answer the following questions: (1) whether increased vessel density translates into improvements in blood flow in the hemodynamically compromised brain; (2) how VEGF influences brain plasticity and contributes to motor and nonmotor recovery; (3) what are the actions of VEGF not only in young animals with preserved vasculature, on which previous studies have been conducted, but also in aged animals and in animals with preexisting atherosclerosis; and (4) whether the effects of VEGF can be mimicked by pharmacological compounds or by cell-based therapies. Only on the basis of such information can more definite conclusions be made with regard to whether the translation of therapeutic angiogenesis into clinics is promising.

Increased Blood–Brain Barrier Permeability and Brain Edema After Focal Cerebral Ischemia Induced by Hyperlipidemia: Role of Lipid Peroxidation and Calpain-1/2, Matrix Metalloproteinase-2/9, and RhoA Overactivation

Background and Purpose— Hyperlipidemia is a highly prevalent risk factor for ischemic stroke. Its impact on brain injury and blood–brain barrier permeability, so far, has not been assessed in animal models of ischemic stroke. Methods— Wild-type and apolipoprotein E−/− mice, fed with normal or cholesterol-rich high-fat food, were subjected to 30 minutes of middle cerebral artery occlusion. Ischemic injury, brain edema, IgG extravasation, lipid peroxidation, calpain-1/2, matrix metalloproteinase-2/9, and RhoA activation, and occludin expression were evaluated 24 hours after reperfusion. Results— Cholesterol-rich food, but not apolipoprotein E deficiency, increased IgG extravasation and brain edema without influencing infarct area and the density of DNA fragmented cells. Increased lipid peroxidation and low-density lipoprotein oxidation were noticed in the brain of hyperlipidemic mice and were associated with increased activation of calpain-1/2 and matrix metalloproteinase-2/9, overactivation of RhoA and its guanine exchange factor leukemia-associated guanine exchange factor , and downregulation of the tight junction protein occludin in cerebral microvessels. Conclusions— That postischemic blood–brain barrier permeability and brain edema are increased during hyperlipidemia points toward the importance of the recognition and adequate treatment of this highly prevalent condition. Translational studies should more adequately mimic risk factors prevalent in human stroke.

Combination of Tissue-Plasminogen Activator With Erythropoietin Induces Blood–Brain Barrier Permeability, Extracellular Matrix Disaggregation, and DNA Fragmentation After Focal Cerebral Ischemia in Mice

Background and Purpose— After 1 clinical study in which recombinant erythropoietin (EPO) protected against ischemic stroke and improved clinical outcome, the German multicenter EPO trial recently reported increased mortality in stroke patients receiving EPO after tissue-plasminogen activator (t-PA)-induced thrombolysis. The reasons for the adverse effects of EPO in t-PA–treated patients are unknown. Methods— Mice were submitted to 90 minutes of middle cerebral artery occlusion. Immediately after reperfusion, animals were treated with normal saline or t-PA (10 mg/kg). Animals subsequently received injections of normal saline or EPO that were administered after reperfusion and 12 hours later (2500 IU/kg each). Ischemic injury and brain edema were analyzed at 24 hours after reperfusion by cresyl violet staining and terminal transferase biotinylated-dUTP nick end labeling. Blood–brain barrier integrity was assessed by histochemistry for extravasated serum IgG. Matrix metalloproteinase activity was evaluated by gelatinase zymography. Results— EPO did not influence ischemic infarct size but reduced brain swelling. This effect was abolished by t-PA, which exacerbated serum IgG extravasation in ischemic tissue. Gelatinase zymographies revealed that EPO promoted matrix metalloproteinase-9 activity that was markedly elevated by t-PA. Add-on treatment with t-PA increased the density of DNA-fragmented cells in ischemic tissue of EPO-treated, but not vehicle-treated, mice. Conclusions— Our data demonstrate a hitherto unknown interaction of t-PA with EPO at the blood–brain interface, ie, promotion of vascular permeability and extracellular matrix breakdown, which may account for the unfavorable actions of EPO in t-PA–treated patients. After t-PA–induced thrombolysis, EPO may not be suitable as stroke treatment.

Vascular Endothelial Growth Factor Promotes Pericyte Coverage of Brain Capillaries, Improves Cerebral Blood Flow During Subsequent Focal Cerebral Ischemia, and Preserves the Metabolic Penumbra

Background and Purpose— Therapeutic angiogenesis aims at improving cerebral blood flow by amplification of vascular sprouting, thus promoting tissue survival under conditions of subsequent ischemia. It remains unknown whether induced angiogenesis leads to the formation of functional vessels that indeed result in hemodynamic improvements. Observations of hemodynamic steal phenomena and disturbed neurovascular integrity after vascular endothelial growth factor delivery questioned the concept of therapeutic angiogenesis. Methods— Mice were treated with recombinant human vascular endothelial growth factor (0.02 &mgr;g/d; intracerebroventricular) for 3 to 21 days and subsequently exposed to 90-minute middle cerebral artery occlusion. Angiogenesis, histological brain injury, IgG extravasation, cerebral blood flow, protein synthesis and energy state, and pericyte coverage on brain capillaries were evaluated in a multiparametric approach combining histochemical, autoradiographic, and regional bioluminescence techniques. Results— Vascular endothelial growth factor increased brain capillary density within 10 days and reduced infarct volume and inflammation after subsequent middle cerebral artery occlusion, and, when delivered for prolonged periods of 21 days, enhanced postischemic blood–brain barrier integrity. Increased cerebral blood flow was noted in ischemic brain areas exhibiting enhanced angiogenesis and was associated with preservation of the metabolic penumbra, defined as brain tissue in which protein synthesis has been suppressed but ATP preserved. Vascular endothelial growth factor enhanced pericyte coverage of brain endothelial cells via mechanisms involving increased N-cadherin expression on cerebral microvessels. Conclusions— That cerebral blood flow is increased during subsequent ischemic episodes, leading to the stabilization of cerebral energy state, fosters hope that by promoting new vessel formation brain tissue survival may be improved.

Effects of vascular endothelial growth factor in ischemic stroke

Vascular endothelial growth factor (VEGF) is a pleiotropic growth factor that is crucially involved in neurovascular remodeling in the ischemic brain. VEGF promotes angiogenesis, protects ischemic neurons from injury, has potent anti‐inflammatory actions, and promotes brain plasticity, in addition to enhancing the recruitment and proliferation of neural precursor cells. These broad actions make VEGF interesting as a model molecule that allows understanding endogenous responses of the brain to injuries. However, several studies indicate that the route and timing of VEGF administration are crucial for the effects of VEGF on ischemic brain tissue. Hence, systemic VEGF delivery in the very acute stroke phase may exacerbate brain damage because of the promotion of blood–brain barrier breakdown that inevitably accompanies vascular growth. Future studies aimed at the promotion of neurovascular remodeling in ischemic stroke should carefully take into consideration pleiotropic actions of angiogenic growth factors beyond vascular growth.

Hyperlipidemia Attenuates Vascular Endothelial Growth Factor–Induced Angiogenesis, Impairs Cerebral Blood Flow, and Disturbs Stroke Recovery via Decreased Pericyte Coverage of Brain Endothelial Cells

Objective—Therapeutic angiogenesis aims at the promotion of vascular growth, usually under conditions of atherosclerosis. It was unknown how hyperlipidemia, a risk factor that is closely associated with atherosclerosis of brain vessels in humans, influences vascular endothelial growth factor–induced angiogenesis and stroke recovery. Approach and Results—Wild-type and apolipoprotein-E (ApoE)−/− mice were kept on regular or cholesterol-rich diet for mimicking different severities of hyperlipidemia. Mice were treated intracerebroventricularly with recombinant human vascular endothelial growth factor for 21 days (0.02 µg/d) and subsequently subjected to 90-minute middle cerebral artery occlusion followed by 1 or 24 hours of reperfusion. Histochemical, autoradiographic, and regional bioluminescence techniques were used to evaluate effects of blood lipids on postischemic angiogenesis, histopathologic brain injury, cerebral blood flow, protein synthesis and energy state, and pericyte coverage of brain endothelial cells. Hyperlipidemia dose-dependently attenuated vascular endothelial growth factor–induced capillary formation and pericyte coverage of brain endothelial cells, abolishing the improvement of cerebral blood flow during subsequent stroke, resulting in the loss of the metabolic penumbra and increased brain infarction. The enhanced angiogenesis after vascular endothelial growth factor treatment was accompanied by increased expression of the adhesion protein N-cadherin, which mediates endothelial-pericytic interactions, in ischemic brain microvessels of wild-type mice on regular diet that was blunted in wild-type mice on Western diet and ApoE−/− mice on either diet. Conclusions—The compromised vessel formation and hemodynamics question the concept of therapeutic angiogenesis in ischemic stroke where hyperlipidemia is highly prevalent.

Transduction of Neural Precursor Cells with TAT‐Heat Shock Protein 70 Chaperone: Therapeutic Potential Against Ischemic Stroke after Intrastriatal and Systemic Transplantation

Novel therapeutic concepts against cerebral ischemia focus on cell‐based therapies in order to overcome some of the side effects of thrombolytic therapy. However, cell‐based therapies are hampered because of restricted understanding regarding optimal cell transplantation routes and due to low survival rates of grafted cells. We therefore transplanted adult green fluorescence protein positive neural precursor cells (NPCs) either intravenously (systemic) or intrastriatally (intracerebrally) 6 hours after stroke in mice. To enhance survival of NPCs, cells were in vitro protein‐transduced with TAT‐heat shock protein 70 (Hsp70) before transplantation followed by a systematic analysis of brain injury and underlying mechanisms depending on cell delivery routes. Transduction of NPCs with TAT‐Hsp70 resulted in increased intracerebral numbers of grafted NPCs after intracerebral but not after systemic transplantation. Whereas systemic delivery of either native or transduced NPCs yielded sustained neuroprotection and induced neurological recovery, only TAT‐Hsp70‐transduced NPCs prevented secondary neuronal degeneration after intracerebral delivery that was associated with enhanced functional outcome. Furthermore, intracerebral transplantation of TAT‐Hsp70‐transduced NPCs enhanced postischemic neurogenesis and induced sustained high levels of brain‐derived neurotrophic factor, glial cell line‐derived neurotrophic factor, and vascular endothelial growth factor in vivo. Neuroprotection after intracerebral cell delivery correlated with the amount of surviving NPCs. On the contrary, systemic delivery of NPCs mediated acute neuroprotection via stabilization of the blood‐brain‐barrier, concomitant with reduced activation of matrix metalloprotease 9 and decreased formation of reactive oxygen species. Our findings imply two different mechanisms of action of intracerebrally and systemically transplanted NPCs, indicating that systemic NPC delivery might be more feasible for translational stroke concepts, lacking a need of in vitro manipulation of NPCs to induce long‐term neuroprotection. STEM CELLS2012;30:1297–1310

Vascular endothelial growth factor induces contralesional corticobulbar plasticity and functional neurological recovery in the ischemic brain

Erratum to: Acta Neuropathol (2012) 123:273–284. DOI 10.1007/s00401‑011‑0914‑z. The authors would like to correct Fig. 3 of the original manuscript, since the image in Fig. 3b does not correspond to a VEGF treated animal. Corrected Fig. 3 is shown below. We apologize for this mistake.

Acute hepatocyte growth factor treatment induces long-term neuroprotection and stroke recovery via mechanisms involving neural precursor cell proliferation and differentiation

Hepatocyte growth factor (HGF) is an interesting candidate for acute stroke treatment as shown by continuous infusion or gene delivery protocols. However, little is known about HGF-mediated long-term effects. The present study therefore analyzed long-term effects of an acute intrastriatal HGF treatment (5 μg) after a 45-minute stroke, with regard to brain injury and neurologic recovery. Hepatocyte growth factor induced long-term neuroprotection as assessed by infarct volume and neuronal cell death analysis for as long as 4 weeks after stroke, which was associated with sustained neurologic recovery as evidenced by corner-turn and tight-rope tests. Analyzing underlying mechanisms of HGF-induced sustained neuroprotection, enhanced cell proliferation followed by increased neuronal differentiation of neural precursor cells (NPCs) was observed in the ischemic striatum of HGF-treated mice, which persisted for up to 4 weeks. In line with this, HGF promoted neurosphere formation as well as proliferation of NPC and decreased caspase-3-dependent hypoxic injury in vitro. Preservation of blood—brain barrier integrity 24 hours after stroke was furthermore noticed in animals receiving HGF, which was associated with the inhibition of matrix metalloproteases (MMP)-2 and MMP-9 at 4 and 24 hours, respectively. We suggest that sustained recruitment of proliferating cells together with improved neurovascular remodeling provides an explanation for HGF-induced long-term neuroprotection.

Intracerebroventricularly delivered VEGF promotes contralesional corticorubral plasticity after focal cerebral ischemia via mechanisms involving anti-inflammatory actions

Vascular endothelial growth factor (VEGF) has potent angiogenic and neuroprotective effects in the ischemic brain. Its effect on axonal plasticity and neurological recovery in the post-acute stroke phase was unknown. Using behavioral tests combined with anterograde tract tracing studies and with immunohistochemical and molecular biological experiments, we examined effects of a delayed i.c.v. delivery of recombinant human VEGF(165), starting 3 days after stroke, on functional neurological recovery, corticorubral plasticity and inflammatory brain responses in mice submitted to 30 min of middle cerebral artery occlusion. We herein show that the slowly progressive functional improvements of motor grip strength and coordination, which are induced by VEGF, are accompanied by enhanced sprouting of contralesional corticorubral fibres that branched off the pyramidal tract in order to cross the midline and innervate the ipsilesional parvocellular red nucleus. Infiltrates of CD45+ leukocytes were noticed in the ischemic striatum of vehicle-treated mice that closely corresponded to areas exhibiting Iba-1+ activated microglia. VEGF attenuated the CD45+ leukocyte infiltrates at 14 but not 30 days post ischemia and diminished the microglial activation. Notably, the VEGF-induced anti-inflammatory effect of VEGF was associated with a downregulation of a broad set of inflammatory cytokines and chemokines in both brain hemispheres. These data suggest a link between VEGFs immunosuppressive and plasticity-promoting actions that may be important for successful brain remodeling. Accordingly, growth factors with anti-inflammatory action may be promising therapeutics in the post-acute stroke phase.