Stem Cell Factor in Combination With Granulocyte Colony-Stimulating Factor Protects the Brain From Capillary Thrombosis-Induced Ischemic Neuron Loss in a Mouse Model of CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarct and leukoencephalopathy (CADASIL) is a Notch3 mutation-induced cerebral small vessel disease, leading to recurrent ischemic stroke and vascular dementia. There is currently no treatment that can stop or delay CADASIL progression. We have demonstrated the efficacy of treatment with combined stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) (SCF+G-CSF) in reducing cerebral small vessel thrombosis in a TgNotch3R90C mouse model of CADASIL. However, it remains unknown whether SCF+G-CSF treatment protects neurons from microvascular thrombosis-induced ischemic damage. Using bone marrow transplantation to track thrombosis, we observed that capillary thrombosis was widely distributed in the cortex, striatum and hippocampus of 22-month-old TgNotch3R90C mice. However, the capillary thrombosis mainly occurred in the cortex. Neuron loss was seen in the area next to the thrombotic capillaries, and severe neuron loss was found in the areas adjacent to the thrombotic capillaries with bifurcations. SCF+G-CSF repeated treatment significantly attenuated neuron loss in the areas next to the thrombotic capillaries in the cortex of the 22-month-old TgNotch3R90C mice. Neuron loss caused by capillary thrombosis in the cerebral cortex may play a crucial role in the pathogenesis of CADASIL. SCF+G-CSF treatment ameliorates the capillary thrombosis-induced ischemic neuron loss in TgNotch3R90C mice. This study provides new insight into the understanding of CADASIL progression and therapeutic potential of SCF+G-CSF in neuroprotection under microvascular ischemia in CADASIL.

Stem Cell Factor in Combination with Granulocyte Colony-Stimulating Factor reduces Cerebral Capillary Thrombosis in a Mouse Model of CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) is a cerebral small vascular disease caused by NOTCH3 mutation-induced vascular smooth muscle cell (VSMC) degeneration, leading to ischemic stroke and vascular dementia. Our previous study has demonstrated that repeated treatment with a combination of stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) reduces VSMC degeneration and cerebral endothelial cell (EC) damage and improves cognitive function in a mouse model of CADASIL (TgNotch3R90C). This study aimed to determine whether cerebral thrombosis occurs in TgNotch3R90C mice and whether repeated SCF+G-CSF treatment reduces cerebral thrombosis in TgNotch3R90C mice. Using the approaches of bone marrow transplantation to track bone marrow-derived cells and confocal imaging, we observed bone marrow-derived blood cell occlusion in cerebral small vessels and capillaries (thrombosis). Most thrombosis occurred in the cerebral capillaries (93% of total occluded vessels), and the thrombosis showed an increased frequency in the regions of capillary bifurcation. Degenerated capillary ECs were seen inside and surrounding the thrombosis, and the bone marrow-derived ECs were also found next to the thrombosis. IgG extravasation was seen in and next to the areas of thrombosis. SCF+G-CSF treatment significantly reduced cerebral capillary thrombosis and IgG extravasation. These data suggest that the EC damage is associated with thrombosis and blood-brain barrier leakage in the cerebral capillaries under the CADASIL-like condition, whereas SCF+G-CSF treatment diminishes these pathological alterations. This study provides new insight into the involvement of cerebral capillary thrombosis in the development of CADASIL and potential approaches to reduce the thrombosis, which may restrict the pathological progression of CADASIL.

Stem cell factor and granulocyte colony-stimulating factor exhibit therapeutic effects in a mouse model of CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a Notch3 dominant mutation-induced cerebral small vascular disease, is characterized by progressive degeneration of vascular smooth muscle cells (vSMCs) of small arteries in the brain, leading to recurrent ischemic stroke, vascular dementia and death. To date, no treatment can stop or delay the progression of this disease. Herein, we determined the therapeutic effects of stem cell factor (SCF) in combination with granulocyte colony-stimulating factor (G-CSF) (SCF+G-CSF) in a mouse model of CADASIL carrying the human mutant Notch3 gene. SCF+G-CSF was subcutaneously administered for 5 days and repeated 4 times with 1-4 month intervals. We found through water maze testing that SCF+G-CSF treatment improved cognitive function. SCF+G-CSF also attenuated vSMC degeneration in small arteries, increased cerebral blood vascular density, and inhibited apoptosis in CADASIL mice. We also discovered that loss of cerebral capillary endothelial cells and neural stem cells/neural progenitor cells (NSCs/NPCs) occurred in CADASIL mice. SCF+G-CSF treatment inhibited the CADASIL-induced cell loss in the endothelia and NSCs/NPCs and promoted neurogenesis. In an in vitro model of apoptosis, SCF+G-CSF prevented apoptotic cell death in vSMCs through AKT signaling and by inhibiting caspase-3 activity. These data suggest that SCF+G-CSF restricts the pathological progression of CADASIL. This study offers new insights into developing therapeutic strategies for CADASIL.

Novel pathological features and potential therapeutic approaches for CADASIL: insights obtained from a mouse model of CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common condition of hereditary stroke and vascular dementia. CADASIL is caused by Notch3 mutation, leading to progressive degeneration of vascular smooth muscle cells (vSMCs) of the small arteries in the brain. However, the pathogenesis of CADASIL remains largely unknown, and treatment that can stop or delay the progression of CADASIL is not yet available. Using both wild type mice and transgenic mice carrying the human mutant Notch3 gene (CADASIL mice), we have recently characterized the pathological features of CADASIL and determined the therapeutic efficacy of two hematopoietic growth factors, stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) in CADASIL. Our findings have revealed novel pathological changes in the endothelium of cerebral capillaries and in the neural stem cells (NSCs). We have also observed the impairment of cognitive function in CADASIL mice. Moreover, SCF+G-CSF treatment improves cognitive function, inhibits Notch3 mutation-induced vSMC degeneration, cerebral blood bed reduction, cerebral capillary damage, and NSC loss, and increases neurogenesis and angiogenesis. Here we compile an overview of our recently published studies, which provide new insights into understanding the pathogenesis of CADASIL and developing therapeutic strategies for this devastating neurological disease.

Atrial fibrillation detected after acute ischemic stroke: evidence supporting the neurogenic hypothesis.

BACKGROUND: It is unknown whether atrial fibrillation (AF) detected after acute ischemic stroke is caused by neurogenic or cardiogenic mechanisms. Based on the potential damage to the autonomic nervous system, neurogenic mechanisms could be implicated in the pathophysiology of newly diagnosed AF. To test this hypothesis, we developed a mechanistic approach by comparing a prespecified set of indicators in acute ischemic stroke patients with newly diagnosed AF, known AF, and sinus rhythm. METHODS: We prospectively assessed every acute ischemic stroke patient undergoing continuous electrocardiographic monitoring from 2008 through 2011. We compared newly diagnosed AF, known AF, and sinus rhythm patients by using 20 indicators grouped in 4 domains: vascular risk factors, underlying cardiac disease, burden of neurological injury, and in-hospital outcome. RESULTS: We studied 275 acute ischemic stroke patients, 23 with newly diagnosed AF, 64 with known AF, and 188 with sinus rhythm. Patients with newly diagnosed AF had a lower proportion of left atrial enlargement (60.9% versus 91.2%, P=.001), a smaller left atrial area (22.0 versus 26.0 cm2, P=.021), and a higher frequency of insular involvement (30.4% versus 9.5%, P=.017) than participants with known AF. Compared with patients in sinus rhythm, those with newly diagnosed AF had a higher proportion of brain infarcts of 15 mm or more (60.9% versus 37.2%, P=.029) and a higher frequency of insular involvement (30.4% versus 7.3%, P<.001). CONCLUSIONS: The low frequency of underlying cardiac disease and the strikingly high proportion of concurrent strategic insular infarctions in patients with newly diagnosed AF provide additional evidence supporting the role of neurogenic mechanisms in a subset of AF detected after acute ischemic stroke.

Newly diagnosed atrial fibrillation linked to wake-up stroke and TIA: hypothetical implications.

BACKGROUND: Based on the higher frequency of paroxysmal atrial fibrillation during night and early morning hours, we sought to analyze the association between newly diagnosed atrial fibrillation and wake-up ischemic cerebrovascular events. METHODS: We prospectively assessed every acute ischemic stroke and TIA patient admitted to our hospital between 2008 and 2011. We used a forward step-by-step multiple logistic regression analysis to assess the relationship between newly diagnosed atrial fibrillation and wake-up ischemic stroke or TIA, after adjusting for significant covariates. RESULTS: The study population comprised 356 patients, 274 (77.0%) with a diagnosis of acute ischemic stroke and 82 (23.0%) with TIA. A total of 41 (11.5%) of these events occurred during night sleep. A newly diagnosed atrial fibrillation was detected in 27 patients of 272 without known atrial fibrillation (9.9%). We found an independent association between newly diagnosed atrial fibrillation and wake-up ischemic stroke and TIA (odds ratio 3.6, 95% confidence interval 1.2-7.7, p = 0.019). CONCLUSIONS: The odds of detecting a newly diagnosed atrial fibrillation were 3-fold higher among wake-up cerebrovascular events than among non-wake-up events. The significance of this independent association between newly diagnosed atrial fibrillation and wake-up ischemic stroke and TIA and the role of other comorbidities should be investigated in future studies.

The role of stem cell factor and granulocyte-colony stimulating factor in treatment of stroke.

Stroke is a serious cerebrovascular disease that causes high mortality and persistent disability in adults worldwide. Stroke is also an enormous public health problem and a heavy public financial burden in the United States. Treatment for stroke is very limited. Thrombolytic therapy by tissue plasminogen activator (tPA) is the only approved treatment for acute stroke, and no effective treatment is available for chronic stroke. Developing new therapeutic strategies, therefore, is a critical need for stroke treatment. This article summarizes the discovery of new routes of treatment for acute and chronic stroke using two hematopoietic growth factors, stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF). In a study of acute stroke, SCF and G-CSF alone or in combination displays neuroprotective effects in an animal model of stroke. SCF appears to be the optimal treatment for acute stroke as the functional outcome is superior to G-CSF alone or in combination (SCF+G-CSF); however, SCF+G-CSF does show better functional recovery than G-CSF. In a chronic stroke study, the therapeutic effects of SCF and G-CSF alone or in combination appear differently as compared with their effects on the acute stroke. SCF+G-CSF induces stable and long-lasting functional improvement; SCF alone also improves functional outcome but its effectiveness is less than SCF+G-CSF, whereas G-CSF shows no therapeutic effects. Although the mechanism by which SCF+G-CSF repairs the brain in chronic stroke remains poorly understood, our recent findings suggest that the SCF+G-CSF-induced functional improvement in chronic stroke is associated with a contribution to increasing angiogenesis and neurogenesis through bone marrow-derived cells and the direct effects on stimulating neurons to form new neuronal networks. These findings would assist in developing new treatment for stroke. The article presents some promising patents on role of stem cell factor and granulocyte-colony stimulating factor in treatment of stroke.

The combination of stem cell factor and granulocyte-colony stimulating factor for chronic stroke treatment in aged animals.

BACKGROUND: Stroke occurs more frequently in the elderly population and presents the number one leading cause of persistent disability worldwide. Lack of effective treatment to enhance brain repair and improve functional restoration in chronic stroke, the recovery phase of stroke, is a challenging medical problem to be solved in stroke research. Our early study has revealed the therapeutic effects of stem cell factor (SCF) in combination with granulocyte-colony stimulating factor (G-CSF) (SCF+G-CSF) on chronic stroke in young animals. However, whether this treatment is effective and safe to the aged population remains to be determined. METHODS: Cortical brain ischemia was produced in aged C57BL mice or aged spontaneously hypertensive rats. SCF+G-CSF or equal volume of vehicle solution was subcutaneously injected for 7 days beginning at 3-4 months after induction of cortical brain ischemia. Using the approaches of biochemistry assays, flow cytometry, pathology, and evaluation of functional outcome, several doses of SCF+G-CSF have been examined for their safety and efficiency on chronic stroke in aged animals. RESULTS: All tested doses did not show acute or chronic toxicity in the aged animals. Additionally, SCF+G-CSF treatment in chronic stroke of aged animals mobilized bone marrow stem cells and improved functional outcome in a dose-dependent manner. CONCLUSIONS: SCF+G-CSF treatment is a safe and effective approach to chronic stroke in the aged condition. This study provides important information needed for developing a new therapeutic strategy to improve the health of older adults with chronic stroke.

Ultrastructural changes in cerebral capillary pericytes in aged Notch3 mutant transgenic mice.

Pericytes, the specialized vascular smooth muscle cells (VSMCs), play an important role in supporting and maintaining the structure of capillaries. Pericytes show biochemical and physiologic features similar to VSMC, usually containing smooth muscle actin fibers and rich endoplasm reticulum. Studies have indicated that degeneration of VSMCs due to Notch3 mutations is the cause of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). However, it remains unclear whether the Notch3 mutation also affects cerebral cortex capillary pericytes. In this ultrastructural morphologic study, the authors have observed pathological changes in the cerebral cortex capillary pericytes in aged mice that carry human mutant Notch3 genes. The number of abnormal pericytes in the cerebral cortex in Notch3 gene mutant mice was slightly increased when compared to an age-matched control group. Morphologically, the pericytes in the brains of Notch3 gene mutant mice showed more severe cellular injury, such as the presence of damaged mitochondria, secondary lysosomes, and large cytoplasmic vesicles. In addition, morphologic structures related to autophagy were also present in the pericytes of Notch3 gene mutant group. These ultrastructural morphologic alterations suggest that Notch3 mutation precipitates age-related pericytic degeneration that might result in cellular injury and trigger autophagic apoptosis. Microvascular dysfunction due to pericyte degeneration could initiate secondary neurodegenerative changes in brain parenchyma. These findings provide new insight into understanding the role of pericyte degeneration in the phathogenesis of CADASIL disease.

Stem cell factor and granulocyte colony-stimulating factor reduce ß-amyloid deposits in the brains of APP/PS1 transgenic mice.

INTRODUCTION: Alzheimer's disease (AD) is widely recognized as a serious public health problem and heavy financial burden. Currently, there is no treatment that can delay or stop the progressive brain damage in AD. Recently, we demonstrated that stem cell factor (SCF) in combination with granulocyte colony-stimulating factor (G-CSF) (SCF+G-CSF) has therapeutic effects on chronic stroke. The purpose of the present study is to determine whether SCF+G-CSF can reduce the burden of ß-amyloid deposits in a mouse model of AD. METHODS: APP/PS1 transgenic mice were used as the model of AD. To track bone marrow-derived cells in the brain, the bone marrow of the APP/PS1 mice was replaced with the bone marrow from mice expressing green fluorescent protein (GFP). Six weeks after bone marrow transplantation, mice were randomly divided into a saline control group and a SCF+G-CSF-treated group. SCF in combination with G-CSF was administered subcutaneously for 12 days. Circulating bone marrow stem cells (CD117+ cells) were quantified 1 day after the final injection. Nine months after treatment, at the age of 18 months, mice were sacrificed. Brain sections were processed for immunohistochemistry to identify ß-amyloid deposits and GFP expressing bone marrow-derived microglia in the brain. RESULTS: Systemic administration of SCF+G-CSF to APP/PS1 transgenic mice leads to long-term reduction of ß-amyloid deposition in the brain. In addition, we have also observed that the SCF+G-CSF treatment increases circulating bone marrow stem cells and augments bone marrow-derived microglial cells in the brains of APP/PS1 mice. Moreover, SCF+G-CSF treatment results in enhancement of the co-localization of bone marrow-derived microglia and ß-amyloid deposits in the brain. CONCLUSIONS: These data suggest that bone marrow-derived microglia play a role in SCF+G-CSF-induced long-term effects to reduce ß-amyloid deposits. This study provides insights into the contribution of the hematopoeitic growth factors, SCF and G-CSF, to limit ß-amyloid accumulation in AD and may offer a new therapeutic approach for AD.

Brain self-protection: the role of endogenous neural progenitor cells in adult brain after cerebral cortical ischemia.

Convincing evidence has shown that brain ischemia causes the proliferation of neural stem cells/neural progenitor cells (NSCs/NPCs) in both the subventricular zone (SVZ) and the subgranular zone (SGZ) of adult brain. The role of brain ischemia-induced NSC/NPC proliferation, however, has remained unclear. Here we have determined whether brain ischemia-induced amplification of the NSCs/NPCs in adult brain is required for brain self-protection. The approach of intracerebroventricular (ICV) infusion of cytosine arabinoside (Ara-C), an inhibitor for cell proliferation, for the first 7days after brain ischemia was used to block ischemia-induced NSC/NPC proliferation. We observed that ICV infusion of Ara-C caused a complete blockade of NSC/NPC proliferation in the SVZ and a dramatic reduction of NSC/NPC proliferation in the SGZ. Additionally, as a result of the inhibition of ischemia-induced NSC/NPC pool amplification, the number of neurons in the hippocampal CA1 and CA3 was significantly reduced, the infarction size was significantly enlarged, and neurological deficits were significantly worsened after focal brain ischemia. We also found that an NSC/NPC-conditioned medium showed neuroprotective effects in vitro and that adult NSC/NPC-released brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) are required for NSC/NPC-conditioned medium-induced neuroprotection. These data suggest that NSC/NPC-generated trophic factors are neuroprotective and that brain ischemia-triggered NSC/NPC proliferation is crucial for brain protection. This study provides insights into the contribution of endogenous NSCs/NPCs to brain self-protection in adult brain after ischemia injury.

The role of stem cell factor and granulocyte-colony stimulating factor in brain repair during chronic stroke.

Chronic stroke is a highly important but under-investigated scientific problem in neurologic research. We have reported earlier that stem cell factor (SCF) in combination with granulocyte-colony stimulating factor (G-CSF) treatment during chronic stroke improves functional outcomes. Here we have determined the contribution of bone marrow-derived cells in angiogenesis and neurogenesis, which are enhanced by SCF+G-CSF treatment during chronic stroke. Using bone marrow tracking, flow cytometry, 2-photon live brain imaging, and immunohistochemistry, we observed that the levels of circulating bone marrow stem cells (BMSCs) (CD34+/c-kit+) were significantly increased by SCF+G-CSF treatment. In addition, live brain imaging revealed that numerous bone marrow-derived cells migrate into the brain parenchyma in the treated mice. We also found that bone marrow-derived cells, bone marrow-derived endothelial cells, vascular density, and bone marrow-derived neurons were significantly augmented by SCF+G-CSF. It is interesting that, in addition to the increase in bone marrow-derived endothelial cells, the number of bone marrow-derived pericytes was reduced after SCF+G-CSF treatment during chronic stroke. These data suggest that SCF+G-CSF treatment can enhance repair of brain damage during chronic stroke by mobilizing BMSCs, and promoting the contribution of bone marrow-derived cells to angiogenesis and neurogenesis.