Nienwen Chow

Role of the MEOX2 homeobox gene in neurovascular dysfunction in Alzheimer disease.

Neurovascular dysfunction substantially contributes to Alzheimer disease. Here, we show that transcriptional profiling of human brain endothelial cells (BECs) defines a subset of genes whose expression is age-independent but is considerably altered in Alzheimer disease, including the homeobox gene MEOX2 (also known as GAX), a regulator of vascular differentiation, whose expression is low in Alzheimer disease. By using viral-mediated MEOX2 gene silencing and transfer, we show that restoring expression of the protein it encodes, GAX, in BECs from individuals with Alzheimer disease stimulates angiogenesis, transcriptionally suppresses AFX1 forkhead transcription factor–mediated apoptosis and increases the levels of a major amyloid-β peptide (Aβ) clearance receptor, the low-density lipoprotein receptor–related protein 1 (LRP), at the blood-brain barrier. In mice, deletion of Meox2 (also known as Gax) results in reductions in brain capillary density and resting cerebral blood flow, loss of the angiogenic response to hypoxia in the brain and an impaired Aβ efflux from brain caused by reduced LRP levels. The link of MEOX2 to neurovascular dysfunction in Alzheimer disease provides new mechanistic and therapeutic insights into this illness.

SRF and myocardin regulate LRP-mediated amyloid-beta clearance in brain vascular cells.

Amyloid β-peptide (Aβ) deposition in cerebral vessels contributes to cerebral amyloid angiopathy (CAA) in Alzheimers disease (AD). Here, we report that in AD patients and two mouse models of AD, overexpression of serum response factor (SRF) and myocardin (MYOCD) in cerebral vascular smooth muscle cells (VSMCs) generates an Aβ non-clearing VSMC phenotype through transactivation of sterol regulatory element binding protein-2, which downregulates low density lipoprotein receptor-related protein-1, a key Aβ clearance receptor. Hypoxia stimulated SRF/MYOCD expression in human cerebral VSMCs and in animal models of AD. We suggest that SRF and MYOCD function as a transcriptional switch, controlling Aβ cerebrovascular clearance and progression of AD.

Serum response factor and myocardin mediate arterial hypercontractility and cerebral blood flow dysregulation in Alzheimer's phenotype

Cerebral angiopathy contributes to cognitive decline and dementia in Alzheimers disease (AD) through cerebral blood flow (CBF) reductions and dysregulation. We report vascular smooth muscle cells (VSMC) in small pial and intracerebral arteries, which are critical for CBF regulation, express in AD high levels of serum response factor (SRF) and myocardin (MYOCD), two interacting transcription factors that orchestrate a VSMC-differentiated phenotype. Consistent with this finding, AD VSMC overexpressed several SRF-MYOCD-regulated contractile proteins and exhibited a hypercontractile phenotype. MYOCD overexpression in control human cerebral VSMC induced an AD-like hypercontractile phenotype and diminished both endothelial-dependent and -independent relaxation in the mouse aorta ex vivo. In contrast, silencing SRF normalized contractile protein content and reversed a hypercontractile phenotype in AD VSMC. MYOCD in vivo gene transfer to mouse pial arteries increased contractile protein content and diminished CBF responses produced by brain activation in wild-type mice and in two AD models, the Dutch/Iowa/Swedish triple mutant human amyloid β-peptide (Aβ)-precursor protein (APP)- expressing mice and APPsw+/− mice. Silencing Srf had the opposite effect. Expression of SRF did not change in VSMC subjected to Alzheimers neurotoxin, Aβ. Thus, SRF-MYOCD overexpression in small cerebral arteries appears to initiate independently of Aβ a pathogenic pathway mediating arterial hypercontractility and CBF dysregulation, which are associated with Alzheimers dementia.

Activated protein C therapy slows ALS-like disease in mice by transcriptionally inhibiting SOD1 in motor neurons and microglia cells

Activated protein C (APC) is a signaling protease with anticoagulant activity. Here, we have used mice expressing a mutation in superoxide dismutase-1 (SOD1) that is linked to amyotrophic lateral sclerosis (ALS) to show that administration of APC or APC analogs with reduced anticoagulant activity after disease onset slows disease progression and extends survival. A proteolytically inactive form of APC with reduced anticoagulant activity provided no benefit. APC crossed the blood-spinal cord barrier in mice via endothelial protein C receptor. When administered after disease onset, APC eliminated leakage of hemoglobin-derived products across the blood-spinal cord barrier and delayed microglial activation. In microvessels, motor neurons, and microglial cells from SOD1-mutant mice and in cultured neuronal cells, APC transcriptionally downregulated SOD1. Inhibition of SOD1 synthesis in neuronal cells by APC required protease-activated receptor-1 (PAR1) and PAR3, which inhibited nuclear transport of the Sp1 transcription factor. Diminished mutant SOD1 synthesis by selective gene excision within endothelial cells did not alter disease progression, which suggests that diminished mutant SOD1 synthesis in other cells, including motor neurons and microglia, caused the APC-mediated slowing of disease. The delayed disease progression in mice after APC administration suggests that this approach may be of benefit to patients with familial, and possibly sporadic, ALS.

Neurovascular Pathways and Alzheimer Amyloid β‐peptide

According to the prevailing amyloid cascade hypothesis, the onset and progression of a chronic neurodegenerative condition in Alzheimer disease (AD) is initiated by the amyloid β‐peptide (Aβ) accumulation in brain and consequent neuronal toxicity. Recent emphasis on co‐morbidity of AD and cerebrovascular disease and the recognition that cerebrovascular dysregulation is an important feature of AD, has shed new light on neurovascular dysfunction as a possible contributor to cognitive decline and Alzheimer neurodegeneration. In the same time, this association has raised a question as to whether there is a causal relationship between cerebrovascular dysregulation and Aβinitiated pathology, and whether influencing targets in the neurovasculature may prevent different forms of Aβ brain accumulation and/or lower pre‐existing accumulates in a later stage of the disease. Pathogenic cascades which operate to dissociate normal transport exchanges between central and peripheral pools of Aβ, and decreased vascular competence leading to brain hypoperfusion and impaired Aβ clearance are discussed. We suggest that there is a link between neurovascular dysfunction and elevated brain AB which provides a new scenario for therapeutic interventions to control Alzheimer mental deterioration.

Neuroprotective activities of activated protein C mutant with reduced anticoagulant activity

The anticoagulant activated protein C (APC) protects neurons and endothelium via protease activated receptor (PAR)1, PAR3 and endothelial protein C receptor. APC is neuroprotective in stroke models. Bleeding complications may limit the pharmacologic utility of APC. Here, we compared the 3K3A‐APC mutant with 80% reduced anticoagulant activity and wild‐type (wt)‐APC. Murine 3K3A‐APC compared with wt‐APC protected mouse cortical neurons from N‐methyl‐D‐aspartate‐induced apoptosis with twofold greater efficacy and more potently reduced N‐methyl‐D‐aspartate excitotoxic lesions in vivo. Human 3K3A‐APC protected human brain endothelial cells (BECs) from oxygen/glucose deprivation with 1.7‐fold greater efficacy than wt‐APC. 3K3A‐APC neuronal protection required PAR1 and PAR3, as shown by using PAR‐specific blocking antibodies and PAR1‐ and PAR3‐deficient cells and mice. BEC protection required endothelial protein C receptor and PAR1. In neurons and BECs, 3K3A‐APC blocked caspase‐9 and ‐3 activation and induction of p53, and decreased the Bax/Bcl‐2 pro‐apoptotic ratio. After distal middle cerebral artery occlusion (dMCAO) in mice, murine 3K3A‐APC compared with vehicle given 4:00 h after dMCAO improved the functional outcome and reduced the infarction volume by 50% within 3 days. 3K3A‐APC compared with wt‐APC multi‐dosing therapy at 12:00 h, 1, 3, 5 and 7 days after dMCAO significantly improved functional recovery and reduced the infarction volume by 75% and 38%, respectively, within 7 days. The wt‐APC, but not 3K3A‐APC, significantly increased the risk of intracerebral bleeding as indicated by a 50% increase in hemoglobin levels in the ischemic hemisphere. Thus, 3K3A‐APC offers a new approach for safer and more efficacious treatments of neurodegenerative disorders and stroke with APC.

An Activated Protein C Analog With Reduced Anticoagulant Activity Extends the Therapeutic Window of Tissue Plasminogen Activator for Ischemic Stroke in Rodents

Background and Purpose— Tissue plasminogen activator (tPA) is the only approved therapy for acute ischemic stroke. However, tPA has a brief therapeutic window. Its side effects include intracerebral bleeding and neurotoxicity. Therefore, a combination therapy with tPA and agents that can extend the therapeutic window of tPA and/or counteract its side effects are warranted. Here, we studied whether 3K3A-APC, a neuroprotective analog of activated protein C with reduced anticoagulant activity, can enhance the therapeutic effects of tPA in models of ischemic stroke in rodents. Methods— Human recombinant tPA (10 mg/kg), alone or in combination with human recombinant 3K3A-APC (2 mg/kg), was administered intravenously 4 hours after proximal or distal transient middle cerebral artery occlusion in mice and embolic stroke in rats. The 3K3A-APC was additionally administered for 3 to 4 consecutive days after stroke. The neuropathological and neurological analyses were performed at 1 to 7 days after stroke. Results— In all models, tPA alone had no effects on the infarct volume or behavior (ie, neurological score, foot-fault, forelimb asymmetry, adhesive removal) compared with vehicle. The tPA and 3K3A-APC combination therapy reduced the infarct volume 24 hours and 7 days after proximal or distal transient middle cerebral artery occlusion in mice and 7 days after embolic stroke in rats by 65%, 63%, and 52%, respectively, significantly (P<0.05) improved behavior and eliminated tPA-induced intracerebral microhemorrhages. Conclusions— The 3K3A-APC extends the therapeutic window of tPA for ischemic stroke in rodents. Therefore, this combination therapy also should be considered for treating stroke in humans.

Differential Neuroprotection and Risk for Bleeding From Activated Protein C With Varying Degrees of Anticoagulant Activity

BACKGROUND AND PURPOSE Activated protein C (APC), a protease with anticoagulant and cytoprotective activities, protects neurons and endothelium from ischemic injury. Drotrecogin-alfa activated, a hyperanticoagulant form of human recombinant APC, is currently being studied in patients with ischemic stroke. How changes in APC anticoagulant activity influence APCs neuroprotection and risk for bleeding is not clear. METHODS We used neuronal and brain endothelial cell injury models and middle cerebral artery occlusion in mice to compare efficacy and safety of drotrecogin-alfa activated and human 3K3A-APC, an APC nonanticoagulant mutant. RESULTS Drotrecogin-alfa activated and 3K3A-APC exhibited 148% and 10% of plasma-derived APCs anticoagulant activity and differ in the carbohydrate content. 3K3A-APC protected mouse neurons from N-methyl-d-aspartate-induced apoptosis and human brain endothelial cell from oxygen-glucose deprivation with 1.8- and 3.1-fold greater efficacy than drotrecogin-alfa activated. Given 5 minutes before transient middle cerebral artery occlusion, 3K3A-APC and drotrecogin-alfa activated (0.5 and 2 mg/kg intravenously) reduced comparably and dose-dependently the infarction lesion up to 85%. 3K3A-APC, but not drotrecogin-alfa activated, improved neurological score dose-dependently (P<0.05). 3K3A-APC did not cause bleeding. In contrast, drotrecogin-alfa activated dose-dependently increased hemoglobin content in postischemic brain. After permanent middle cerebral artery occlusion, 3K3A-APC multidose therapy (1 mg/kg intravenously at 12 hours and 1, 3, 5, and 7 days) improved functional recovery and reduced infarction by 60% with no risk for bleeding, whereas drotrecogin-alfa activated increased hemoglobin deposition in the postischemic brain and showed relatively modest neuroprotection. CONCLUSIONS Nonanticoagulant 3K3A-APC exhibits greater neuroprotective efficacy with no risk for bleeding compared with drotrecogin-alfa activated, a hyperanticoagulant form of APC.

An activated protein C analog stimulates neuronal production by human neural progenitor cells via a PAR1-PAR3-S1PR1-Akt pathway.

Activated protein C (APC) is a protease with anticoagulant and cell-signaling activities. In the CNS, APC and its analogs with reduced anticoagulant activity but preserved cell signaling activities, such as 3K3A-APC, exert neuroprotective, vasculoprotective, and anti-inflammatory effects. Murine APC promotes subependymal neurogenesis in rodents in vivo after ischemic and traumatic brain injury. Whether human APC can influence neuronal production from resident progenitor cells in humans is unknown. Here we show that 3K3A-APC, but not S360A-APC (an enzymatically inactive analog of APC), stimulates neuronal mitogenesis and differentiation from fetal human neural stem and progenitor cells (NPCs). The effects of 3K3A-APC on proliferation and differentiation were comparable to those obtained with fibroblast growth factor and brain-derived growth factor, respectively. Its promoting effect on neuronal differentiation was accompanied by inhibition of astroglial differentiation. In addition, 3K3A-APC exerted modest anti-apoptotic effects during neuronal production. These effects appeared to be mediated through specific protease activated receptors (PARs) and sphingosine-1-phosphate receptors (S1PRs), in that siRNA-mediated inhibition of PARs 1–4 and S1PRs 1–5 revealed that PAR1, PAR3, and S1PR1 are required for the neurogenic effects of 3K3A-APC. 3K3A-APC activated Akt, a downstream target of S1PR1, which was inhibited by S1PR1, PAR1, and PAR3 silencing. Adenoviral transduction of NPCs with a kinase-defective Akt mutant abolished the effects of 3K3A-APC on NPCs, confirming a key role of Akt activation in 3K3A-APC-mediated neurogenesis. Therefore, APC and its pharmacological analogs, by influencing PAR and S1PR signals in resident neural progenitor cells, may be potent modulators of both development and repair in the human CNS.

Activated Protein C Analog Protects From Ischemic Stroke and Extends the Therapeutic Window of Tissue-Type Plasminogen Activator in Aged Female Mice and Hypertensive Rats

Background and Purpose— 3K3A-activated protein C (APC) protects young, healthy male rodents after ischemic stroke. 3K3A-APC is currently under development as a neuroprotectant for acute ischemic stroke in humans. Stroke Therapy Academic Industry Roundtable recommends that after initial studies in young, healthy male animals, further studies should be performed in females, aged animals, and animals with comorbid conditions. Here, we studied the effects of delayed 3KA-APC therapy alone and with tissue-type plasminogen activator (tPA) in aged female mice and spontaneously hypertensive rats. Methods— We used Stroke Therapy Academic Industry Roundtable recommendations for ensuring good scientific inquiry. Murine recombinant 3K3A-APC (0.2 mg/kg) alone or with recombinant tPA (10 mg/kg) was given intravenously 4 hours after transient middle cerebral artery occlusion in aged female mice and rats and after embolic stroke in spontaneously hypertensive rat. 3K3A-APC was additionally administered within 3 to 7 days after stroke. The neuropathological analysis and neurological scores, foot-fault, forelimb asymmetry, and adhesive removal tests were performed within 7 and 28 days of stroke. Results— In all models, tPA alone had no effects on the infarct volume or behavior. 3K3A-APC alone or with tPA reduced the infarct volume 7 days after the middle cerebral artery occlusion in aged female mice and embolic stroke in spontaneously hypertensive rat by 62% to 66% and 50% to 53%, respectively, significantly improved (P<0.05) behavior, and eliminated tPA-induced intracerebral microhemorrhages. In aged female mice, 3K3A-APC was protective within 4 weeks of stroke. Conclusions— 3K3A-APC protects from ischemic stroke and extends the therapeutic window of tPA in aged female mice and in spontaneously hypertensive rat with a comorbid condition.