Stem cell factor and granulocyte colony-stimulating factor promote brain repair and improve cognitive function through VEGF-A in a mouse model of CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarct and leukoencephalopathy (CADASIL) is a cerebral small vascular disease caused by NOTCH3 gene mutation in vascular smooth muscle cells (VSMCs), leading to ischemic stroke and vascular dementia. To date, the pathogenesis of CADASIL remains poorly understood, and there is no treatment that can slow the progression of CADASIL. Using a transgenic mouse model of CADASIL (TgNotch3R90C), this study reveals novel findings for understanding CADASIL pathogenesis that decreased cerebral vascular endothelial growth factor (VEGF/VEGF-A) is linked to reduced cerebral blood vessel density. Reduced endothelial cell (EC) proliferation and angiogenesis are seen in TgNotch3R90C mouse brain-isolated ECs. Decreased dendrites, axons, and synapses in the somatosensory and motor cortex layer 2/3 and in the hippocampal CA1, and reduced neurogenesis in both the subventricular zone and subgranular zone occur in 15-month-old TgNotch3R90C mice. These reductions in neuron structures, synapses, and neurogenesis are significantly correlated to decreased cerebral vasculature in the corresponding areas. Impaired spatial learning and memory in TgNotch3R90C mice are significantly correlated with the reduced cerebral vasculature, neuron structures, and synapses. Repeated treatment of stem cell factor and granulocyte colony-stimulating factor (SCF+G-CSF) at 9 and 10 months of age improves cognitive function, increases cerebral VEGF/VEGF-A, restores cerebral vasculature, and enhances regeneration of neuronal structures, synaptogenesis and neurogenesis in TgNotch3R90C mice. Pretreatment with Avastin, an angiogenesis inhibitor by neutralizing VEGF-A, completely eliminates the SCF+G-CSF-enhanced cognitive function, vascular and neuronal structure regeneration, synaptogenesis and neurogenesis in TgNotch3R90C mice. SCF+G-CSF-enhanced EC proliferation and angiogenesis in TgNotch3R90C mouse brain-isolated ECs are also blocked by Avastin pretreatment. These data suggest that SCF+G-CSF treatment may repair Notch3R90C mutation-damaged brain through the VEGF-A-mediated angiogenesis. This study provides novel insight into the involvement of VEGF/VEGF-A in the pathogenesis of CADASIL and sheds light on the mechanism underlying the SCF+G-CSF-enhanced brain repair in CADASIL.

Genetic susceptibility and causal pathway analysis of eye disorders coexisting in multiple sclerosis.

Introduction: The comorbidity of optic neuritis with multiple sclerosis has been well recognized. However, the causal association between multiple sclerosis and optic neuritis, as well as other eye disorders, remains incompletely understood. To address these gaps, we investigated the genetically relationship between multiple sclerosis and eye disorders, and explored potential drugs. Methods: In order to elucidate the genetic susceptibility and causal links between multiple sclerosis and eye disorders, we performed two-sample Mendelian randomization analyses to examine the causality between multiple sclerosis and eye disorders. Additionally, causal single-nucleotide polymorphisms were annotated and searched for expression quantitative trait loci data. Pathway enrichment analysis was performed to identify the possible mechanisms responsible for the eye disorders coexisting with multiple sclerosis. Potential therapeutic chemicals were also explored using the Cytoscape. Results: Mendelian randomization analysis revealed that multiple sclerosis increased the incidence of optic neuritis while reducing the likelihood of concurrent of cataract and macular degeneration. Gene Ontology enrichment analysis implicated that lymphocyte proliferation, activation and antigen processing as potential contributors to the pathogenesis of eye disorders coexisting with multiple sclerosis. Furthermore, pharmaceutical agents traditionally employed for allograft rejection exhibited promising therapeutic potential for the eye disorders coexisting with multiple sclerosis. Discussion: Multiple sclerosis genetically contributes to the development of optic neuritis while mitigating the concurrent occurrence of cataract and macular degeneration. Further research is needed to validate these findings and explore additional mechanisms underlying the comorbidity of multiple sclerosis and eye disorders.

A Novel Effect of Id2 in Microglia TNFα Regulation.

Microglia are the most important immune cells in the central nervous system (CNS), which can defend against external pathogens and stimuli. Dysregulation of microglia releases excessive proinflammatory cytokines and leads to neuroinflammation, which is fundamental to the pathophysiology of multiple neurological diseases. However, the molecular mechanisms underlying the regulation of proinflammatory cytokines in microglia are still not well-understood. Here, we identified that inhibitor of DNA binding protein 2 (Id2) was a negative regulator of tumor necrosis factor-α (TNFα) in cultured microglia. Knockdown of Id2 significantly increased the expression of TNFα in microglia, while overexpression of Id2 inhibited TNFα expression. Furthermore, by interacting with the p65 subunit of nuclear factor kappa-B (NF-κB), Id2 suppressed the transcription activation of NF-κB and inhibited TNFα expression. Interestingly, in lipopolysaccharides (LPS)-treated microglia, Id2 increased and underwent a cytoplasmic relocation. Immunoprecipitation and immunostaining results showed that by binding to the LIM domain of Id2, a scaffold protein PDZ and LIM 5 (PDLIM5) involved in the Id2 cytoplasmic relocation, which inactivated Id2 and resulted in higher TNFα expression in LPS-treated microglia. Collectively, our data delineate a novel effect of Id2 on TNFα regulation in microglia, which may shed a light on the proinflammatory cytokines regulating in microglia associated neuroimmune disorders.

Stem Cell Factor and Granulocyte Colony-Stimulating Factor Promote Remyelination in the Chronic Phase of Severe Traumatic Brain Injury.

Severe traumatic brain injury (TBI) causes long-term disability and death in young adults. White matter is vulnerable to TBI damage. Demyelination is a major pathological change of white matter injury after TBI. Demyelination, which is characterized by myelin sheath disruption and oligodendrocyte cell death, leads to long-term neurological function deficits. Stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) treatments have shown neuroprotective and neurorestorative effects in the subacute and chronic phases of experimental TBI. Our previous study has revealed that combined SCF and G-CSF treatment (SCF + G-CSF) enhances myelin repair in the chronic phase of TBI. However, the long-term effect and mechanism of SCF + G-CSF-enhanced myelin repair remain unclear. In this study, we uncovered persistent and progressive myelin loss in the chronic phase of severe TBI. SCF + G-CSF treatment in the chronic phase of severe TBI enhanced remyelination in the ipsilateral external capsule and striatum. The SCF + G-CSF-enhanced myelin repair is positively correlated with the proliferation of oligodendrocyte progenitor cells in the subventricular zone. These findings reveal the therapeutic potential of SCF + G-CSF in myelin repair in the chronic phase of severe TBI and shed light on the mechanism underlying SCF + G-CSF-enhanced remyelination in chronic TBI.

Stem cell factor and granulocyte colony-stimulating factor promote remyelination in the chronic phase of severe traumatic brain injury.

Severe traumatic brain injury (TBI) causes long-term disability and death in young adults. White matter is vulnerable to TBI damage. Demyelination is a major pathological change of white matter injury after TBI. Demyelination which is characterized by myelin sheath disruption and oligodendrocyte cell death leads to long-term neurological function deficits. Stem cell factor (SCF) and granulocyte colonyâ€"stimulating factor (G-CSF) treatments have shown neuroprotective and neurorestorative effects in the subacute and chronic phases of experimental TBI. Our previous study has revealed that combined SCF and G-CSF treatment (SCF+G-CSF) enhances myelin repair in the chronic phase of TBI. However, the long-term effect and mechanism of SCF+G-CSF-enhanced myelin repair remain unclear. In this study, we uncovered persistent and progressive myelin loss in the chronic phase of severe TBI. SCF+G-CSF treatment in the chronic phase of severe TBI enhanced remyelination in the ipsilateral external capsule and striatum. The SCF+G-CSF-enhanced myelin repair is positively correlated with the proliferation of oligodendrocyte progenitor cells in the subventricular zone. These findings reveal the therapeutic potential of SCF+G-CSF in myelin repair in the chronic phase of severe TBI and shed light on the mechanism underlying SCF+G-CSF-enhanced remyelination in chronic TBI.

Gut microbes exacerbate systemic inflammation and behavior disorders in neurologic disease CADASIL.

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a cerebral small vessel disease that carries mutations in NOTCH3. The clinical manifestations are influenced by genetic and environmental factors that may include gut microbiome. RESULTS: We investigated the fecal metagenome, fecal metabolome, serum metabolome, neurotransmitters, and cytokines in a cohort of 24 CADASIL patients with 28 healthy household controls. The integrated-omics study showed CADASIL patients harbored an altered microbiota composition and functions. The abundance of bacterial coenzyme A, thiamin, and flavin-synthesizing pathways was depleted in patients. Neurotransmitter balance, represented by the glutamate/GABA (4-aminobutanoate) ratio, was disrupted in patients, which was consistent with the increased abundance of two major GABA-consuming bacteria, Megasphaera elsdenii and Eubacterium siraeum. Essential inflammatory cytokines were significantly elevated in patients, accompanied by an increased abundance of bacterial virulence gene homologs. The abundance of patient-enriched Fusobacterium varium positively correlated with the levels of IL-1ß and IL-6. Random forest classification based on gut microbial species, serum cytokines, and neurotransmitters showed high predictivity for CADASIL with AUC = 0.89. Targeted culturomics and mechanisms study further showed that patient-derived F. varium infection caused systemic inflammation and behavior disorder in Notch3R170C/+ mice potentially via induction of caspase-8-dependent noncanonical inflammasome activation in macrophages. CONCLUSION: These findings suggested the potential linkage among the brain-gut-microbe axis in CADASIL. Video Abstract.

SCF + G-CSF treatment in the chronic phase of severe TBI enhances axonal sprouting in the spinal cord and synaptic pruning in the hippocampus.

Traumatic brain injury (TBI) is a major cause of long-term disability in young adults. An evidence-based treatment for TBI recovery, especially in the chronic phase, is not yet available. Using a severe TBI mouse model, we demonstrate that the neurorestorative efficacy of repeated treatments with stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) (SCF + G-CSF) in the chronic phase is superior to SCF + G-CSF single treatment. SCF + G-CSF treatment initiated at 3 months post-TBI enhances contralesional corticospinal tract sprouting into the denervated side of the cervical spinal cord and re-balances the TBI-induced overgrown synapses in the hippocampus by enhancing microglial function of synaptic pruning. These neurorestorative changes are associated with SCF + G-CSF-improved somatosensory-motor function and spatial learning. In the chronic phase of TBI, severe TBI-caused microglial degeneration in the cortex and hippocampus is ameliorated by SCF + G-CSF treatment. These findings reveal the therapeutic potential and possible mechanism of SCF + G-CSF treatment in brain repair during the chronic phase of severe TBI.

Brain-derived CCR5 Contributes to Neuroprotection and Brain Repair after Experimental Stroke.

Chemokine (C-C motif) receptor 5 (CCR5) is expressed not only in the immune cells but also in cerebral cells such as neurons, glia, and vascular cells. Stroke triggers high expression of CCR5 in the brain. However, the role of CCR5 in stroke remains unclear. In this study, using bone marrow chimeras we have determined the involvement of brain-derived or bone marrow-derived CCR5 in neuroprotection and brain repair after experimental stroke. CCR5-/- mice that received either wild-type (WT) or CCR5-/- bone marrow transplantation showed larger infarction sizes than the WT mice that received either WT or CCR5-/- bone marrow transplantation in both the acute (48h) and subacute (2 months) phases after cerebral cortical ischemia, suggesting that the lack of CCR5 in the brain leads to severe brain damage after stroke. However, the lack of CCR5 in the bone marrow-derived cells did not affect infarction size. The impairments of somatosensory-motor function and motor coordination were exacerbated in the mice lacking CCR5 in the brain. At 2 months post-stroke, increased degenerative neurons, decreased dendrites and synapses, decreased Iba1+ microglia/ macrophages, reduced myelination and CNPase+ oligodendrocytes in the peri-infarct cortex were observed in the mice lacking CCR5 in the brain. These pathological changes are significantly correlated with the increased infarction size and exacerbated neurological deficits. These data suggest that brain-derived CCR5 plays a key role in neuroprotection and brain repair in the subacute phase of stroke. This study reveals a novel role of CCR5 in stroke, which sheds new light on post-stroke pathomechanism.

Long-term beneficial effects of hematopoietic growth factors on brain repair in the chronic phase of severe traumatic brain injury.

Severe traumatic brain injury (TBI) is the major cause of long-term, even life-long disability and cognitive impairments in young adults. The lack of therapeutic approaches to improve recovery in the chronic phase of severe TBI is a big challenge to the medical research field. Using a single severe TBI model in young adult mice, this study examined the restorative efficacy of two hematopoietic growth factors, stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF), on brain repair in the chronic phase of TBI. SCF and G-CSF alone or combination (SCF + G-CSF) treatment was administered at 3 months post-TBI. Functional recovery was evaluated by neurobehavioral tests during the period of 21 weeks after treatment. Neuropathology was examined 22 weeks after treatment. We observed that severe TBI caused persistent impairments in spatial learning/memory and somatosensory-motor function, long-term and widespread neuropathology, including dendritic reduction, decrease and overgrowth of axons, over-generated excitatory synapses, and demyelination in the cortex, hippocampus and striatum. SCF, G-CSF, and SCF + G-CSF treatments ameliorated severe TBI-induced widespread neuropathology. SCF + G-CSF treatment showed superior efficacy in improving long-term functional outcome, enhancing neural plasticity, rebalancing neural structure networks disturbed by severe TBI, and promoting remyelination. These novel findings demonstrate the therapeutic potential of SCF and G-CSF in enhancing recovery in the chronic phase of severe TBI .

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.

S100 Calcium-Binding Protein A9 Knockout Contributes to Neuroprotection and Functional Improvement after Traumatic Brain Injury.

S100 calcium-binding protein A9 (S100a9), a proinflammatory protein, has been shown to be involved in the development of neuroinflammatory disorders and neurodegenerative diseases. Upregulation of S100a9 in the brain during acute brain injury has been proposed to be associated with acute neuroinflammation. However, it remains unclear whether eliminating S100a9 expression will show beneficial outcomes after traumatic brain injury (TBI). Using S100a9 knockout mice, this study has demonstrated that S100a9 deletion ameliorates post-TBI anxiety, improves TBI-impaired motor and cognitive function, reduces lesion size, prevents perilesional neuron loss and neurodegeneration, diminishes neuroinflammation and TBI-induced neurogenesis, and enhances perilesional expression of neuroplasticity protein. These findings suggest that S100a9 plays a detrimental role in TBI. Genetic deletion of S100a9 enhances neuroprotection and improves functional outcome after TBI. This study sheds light on the pathological involvement of S100a9 in TBI, which would provide a new therapeutic target to minimize TBI-induced brain damage.

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.

Protein disulfide isomerase-mediated apoptosis and proliferation of vascular smooth muscle cells induced by mechanical stress and advanced glycosylation end products result in diabetic mouse vein graft atherosclerosis.

Protein disulfide isomerase (PDI) involves cell survival and death. Whether PDI mediates mechanical stretch stress (SS) and/or advanced glycosylation end products (AGEs) -triggered simultaneous increases in proliferation and apoptosis of vascular smooth muscle cells (VSMCs) is unknown. Here, we hypothesized that different expression levels of PDI trigger completely opposite cell fates among the different VSMC subtypes. Mouse veins were grafted into carotid arteries of non-diabetic and diabetic mice for 8 weeks; the grafted veins underwent simultaneous increases in proliferation and apoptosis, which triggered vein graft arterializations in non-diabetic or atherosclerosis in diabetic mice. A higher rate of proliferation and apoptosis was seen in the diabetic group. SS and/or AGEs stimulated the quiescent cultured VSMCs, resulting in simultaneous increases in proliferation and apoptosis; they could induce increased PDI activation and expression. Both in vivo and in vitro, the proliferating VSMCs indicated weak co-expression of PDI and SM-α-actin while apoptotic or dead cells showed strong co-expression of both. Either SS or AGEs rapidly upregulated the expression of PDI, NOX1 and ROS, and their combination had synergistic effects. Inhibiting PDI simultaneously suppressed the proliferation and apoptosis of VSMCs, while inhibition of SM-α-actin with cytochalasin D led to increased apoptosis and cleaved caspases-3 but had no effect on proliferation. In conclusion, different expression levels of PDI in VSMCs induced by SS and/or AGEs triggered a simultaneous increase in proliferation and apoptosis, accelerated vein graft arterializations or atherosclerosis, leading us to propose PDI as a novel target for the treatment of vascular remodeling and diseases.

Simultaneous Increases in Proliferation and Apoptosis of Vascular Smooth Muscle Cells Accelerate Diabetic Mouse Venous Atherosclerosis.

AIMS: This study was designed to demonstrate simultaneous increases in proliferation and apoptosis of vascular smooth muscle cells (VSMCs) leading to accelerated vein graft remodeling and to explore the underlying mechanisms. METHODS: Vein grafts were performed in non-diabetic and diabetic mice. The cultured quiescent VSMCs were subjected to mechanical stretch stress (SS) and/or advanced glycosylation end products (AGEs). Harvested vein grafts and treated VSMCs were used to detect cell proliferation, apoptosis, mitogen-activated protein kinases (MAPKs) activation and SM-α-actin expression. RESULTS: Significantly thicker vessel walls and greater increases in proliferation and apoptosis were observed in diabetic vein grafts than those in non-diabetic. Both SS and AGEs were found to induce different activation of three members of MAPKs and simultaneous increases in proliferation and apoptosis of VSMCs, and combined treatment with both had a synergistic effect. VSMCs with strong SM-α-actin expression represented more activated JNKs or p38MAPK, and cell apoptosis, while the cells with weak SM-α-actin expression demonstrated preferential activation of ERKs and cell proliferation. In contrast, inhibition of MAPKs signals triggered significant decreases in VSMC proliferation, and apoptosis. Treatment of the cells with RNA interference of receptor of AGEs (RAGE) also resulted in significant decreases in both proliferation and apoptosis. CONCLUSIONS: Increased pressure-induced SS triggers simultaneous increases in proliferation and apoptosis of VSMCs in the vein grafts leading to vein arterializations, which can be synergistically accelerated by high glucose-induced AGEs resulting in vein graft atherosclerosis. Either SS or AGEs and their combination induce simultaneous increases in proliferation and apoptosis of VSMCs via different activation of three members of MAPKs resulting from different VSMC subtypes classified by SM-α-actin expression levels.

Early administration of tumor necrosis factor-alpha antagonist promotes survival of transplanted neural stem cells and axon myelination after spinal cord injury in rats.

Neural stem cell (NSC) transplantation has been reported to be a leading strategy to stimulate neuroplasticity, repair neuronal loss and promote the morphologic and functional recovery of spinal cord injury (SCI). However, massive death of transplanted NSCs is still a problem, which is considered to be related to a series of pro-inflammatory cytokines that induce apoptosis, extensive demyelination and axonal destruction. Tumor necrosis factor alpha (TNF-α), as one of the major inflammation initiators, contributes to secondary neural cell death. We previously found that the administration of the TNF-α antagonist etanercept during the acute phase of SCI can reduce the apoptosis of neurons and oligodendrocytes. To investigate whether etanercept can suppress transplanted NSC apoptosis and promote NSC survival, axon myelination and functional recovery, we tested the combination strategy of the early administration of etanercept and NSC transplantation. First we observed that etanercept suppressed the TNF-α expression and apoptosis of transplanted NSCs by Western blot, TUNEL and immunofluorescence staining. The Basso, Beattle and Bresnahan scale and motor-evoked potential were used to evaluate functional recovery. The results suggest significantly better recovery after combination therapy. Further, histopathological alterations were evaluated by hematoxylin and eosin staining and Nissl staining. These procedures showed that the early administration of etanercept improved survival of neurons in the ventral horn, restored neural morphology and produced a smaller cavity area. We observed most abundant NF-positive fibers after the combination treatment, indicating that combination therapy retained and promoted neural regeneration. Finally, the early suppression of TNF-α reduced the occurrence of demyelination, and the combination therapy led to more myelinated axons, as shown by electron microscopy. These data suggest that this strategy significantly protected transplanted NSCs via the anti-inflammation and anti-apoptosis effects of etanercept, promoting re-myelination, neural regeneration and locomotor function.

Simvastatin inhibits the additive activation of ERK1/2 and proliferation of rat vascular smooth muscle cells induced by combined mechanical stress and oxLDL through LOX-1 pathway.

Vein grafts interposed into arteries are susceptible to the development of atherosclerosis due to rapid increases in blood pressure. This process is accelerated in patients with hyperlipidemia. The molecular mechanism underlying this process is unknown. In this study, quiescent rat vascular smooth muscle cells (VSMCs) were treated in vitro with mechanical stretch stress (10% elongation) with and without oxLDL (25 µg/ml) in the presence and absence of simvastatin (2.5 µmol/L). The results demonstrate that stretch stress and oxLDL can each induce activation of ERK1/2 and Ki-67 expression in VSMCs, but the peak levels of ERK activation and Ki-67 expression were observed in groups subjected to both stretch stress and oxLDL. Simvastatin was found to inhibit increased ERK activation and Ki-67 expression in VSMCs subjected to stretch stress with or without oxLDL. Mechanically, simvastatin was also found to inhibit increased expression of LOX-1 (a receptor of oxLDL) in VSMCs subjected to stretch stress with or without oxLDL. Knockdown of LOX-1 via small interfering RNAs (siRNA-LOX-1) resulted in obvious inhibition of ERK activation in VSMCs subjected to stretch stress with and without oxLDL. These results suggest that combined stretch stress and oxLDL can additively promote the activation of ERK1/2 leading to accelerated proliferation of VSMCs (e.g. increased Ki-67 expression) via LOX-1 signal pathway. This was found to be partially inhibited by simvastatin. These results may provide important data for the treatment and prevention of hypertension with or without hyperlipidemia.

RAGE mediates accelerated diabetic vein graft atherosclerosis induced by combined mechanical stress and AGEs via synergistic ERK activation.

AIMS/HYPOTHESIS: Diabetes with hypertension rapidly accelerates vascular disease, but the underlying mechanism remains unclear. We evaluated the hypothesis that the receptor of advanced glycation end products (RAGE) might mediate combined signals initiated by diabetes-related AGEs and hypertension-induced mechanical stress as a common molecular sensor. METHODS: In vivo surgical vein grafts created by grafting vena cava segments from C57BL/6J mice into the common carotid arteries of streptozotocin (STZ)-treated and untreated isogenic mice for 4 and 8 weeks were analyzed using morphometric and immunohistochemical techniques. In vitro quiescent mouse vascular smooth muscle cells (VSMCs) with either knockdown or overexpression of RAGE were subjected to cyclic stretching with or without AGEs. Extracellular signal-regulated kinase (ERK) phosphorylation and Ki-67 expression were investigated. RESULTS: Significant increases in neointimal formation, AGE deposition, Ki-67 expression, and RAGE were observed in the vein grafts of STZ-induced diabetic mice. The highest levels of ERK phosphorylation and Ki-67 expression in VSMCs were induced by simultaneous stretch stress and AGE exposure. The synergistic activation of ERKs and Ki-67 in VSMCs was significantly inhibited by siRNA-RAGE treatment and enhanced by over-expression of RAGE. CONCLUSION: RAGE may mediate synergistically increased ERK activation and VSMC proliferation induced by mechanical stretching with and without AGEs. It may serve as a common molecular bridge between the two, accelerating vascular remodeling. This study provides potential drug targets and novel therapeutic strategies for the treatment of vascular diseases resulting from diabetes with hypertension.