Human Brain Microvascular Pericytes Research Articles

Endothelin-1 (ET-1) contributes to senescence and phenotypic changes in brain pericytes in diabetes-mimicking conditions.

Diabetes mediates endothelial dysfunction and increases the risk of Alzheimer's disease and related dementias. Diabetes also dysregulates the ET system. ET-1-mediated constriction of brain microvascular pericytes (BMVPCs) has been shown to contribute to brain hypoperfusion. Cellular senescence, a process that arrests the proliferation of harmful cells and instigates phenotypical changes and proinflammatory responses in endothelial cells that impact their survival and function. Thus, we hypothesized that ET-1 mediates BMVPC senescence and phenotypical changes in diabetes-like conditions. Human BMVPCs were incubated in diabetes-like conditions with or without ET-1 (1 µmol/L) for 3 and 7 days. Hydrogen peroxide (100 µmol/L H2O2) was used as a positive control for senescence and to mimic ischemic conditions. Cells were stained for senescence-associated β-galactosidase or processed for immunoblotting and quantitative real-time PCR analyses. In additional experiments, cells were stimulated with ET-1 in the presence or absence of ETA receptor antagonist BQ-123 (20 μmol/L) or ETB receptor antagonist BQ-788 (20 μmol/L). ET-1 stimulation increased β-galactosidase accumulation which was prevented by BQ-123. ET-1 also increased traditional senescence marker p16 protein and pericyte-specific senescence markers, TGFB1i1, PP1CA, and IGFBP7. Furthermore, ET-1 stimulated contractile protein α-SMA and microglial marker ostepontin in high glucose suggesting a shift toward an ensheathing or microglia-like phenotype. In conclusion, ET-1 triggers senescence, alters ETA and ETB receptors, and causes phenotypical changes in BMVPCs under diabetes-like conditions. These in vitro findings need to be further studied in vivo to establish the role of ETA receptors in the progression of pericyte senescence and phenotypical changes in VCID.

Leptin Promotes Angiogenesis via Pericyte STAT3 Pathway upon Intracerebral Hemorrhage.

Angiogenesis is a vital endogenous brain self-repair processes for neurological recovery after intracerebral hemorrhage (ICH). Increasing evidence suggests that leptin potentiates angiogenesis and plays a beneficial role in stroke. However, the proangiogenic effect of leptin on ICH has not been adequately explored. Moreover, leptin triggers post-ICH angiogenesis through pericyte, an important component of forming new blood vessels, which remains unclear. Here, we reported that exogenous leptin infusion dose-dependent promoted vascular endothelial cells survival and proliferation at chronic stage of ICH mice. Additionally, leptin robustly ameliorated pericytes loss, enhanced pericytes proliferation and migration in ICH mice in vivo, and in ICH human brain microvascular pericytes (HBVPC) in vitro. Notably, we showed that pericytes-derived pro-angiogenic factors were responsible for enhancing the survival, proliferation and tube formation followed leptin treatment in human brain microvascular endothelial cells (HCMEC/D3)/HBVPC co-culture models. Importantly, considerable improvements in neurobehavioral function and hostile microenvironment were observed in leptin treatment ICH mice, indicating that better vascular functionality post ICH improves outcome. Mechanistically, this study unveiled that leptin boost post-ICH angiogenesis potentially through modulation of leptin receptor (leptinR)/Signal Transducer and Activator of Transcription 3 (STAT3) signaling pathway in pericyte. Thus, leptin may be a lucrative option for the treatment of ICH.

GLP-1 receptor nitration contributes to loss of brain pericyte function in a mouse model of diabetes.

We have previously shown that diabetes causes pericyte dysfunction, leading to loss of vascular integrity and vascular cognitive impairment and dementia (VCID). Glucagon-like peptide-1 (GLP-1) receptor agonists (GLP-1 RAs), used in managing type 2 diabetes mellitus, improve the cognitive function of diabetic individuals beyond glycaemic control, yet the mechanism is not fully understood. In the present study, we hypothesise that GLP-1 RAs improve VCID by preventing diabetes-induced pericyte dysfunction. Mice with streptozotocin-induced diabetes and non-diabetic control mice received either saline (NaCl 154 mmol/l) or exendin-4, a GLP-1 RA, through an osmotic pump over 28 days. Vascular integrity was assessed by measuring cerebrovascular neovascularisation indices (vascular density, tortuosity and branching density). Cognitive function was evaluated with Barnes maze and Morris water maze. Human brain microvascular pericytes (HBMPCs), were grown in high glucose (25 mmol/l) and sodium palmitate (200 μmol/l) to mimic diabetic conditions. HBMPCs were treated with/without exendin-4 and assessed for nitrative and oxidative stress, and angiogenic and blood-brain barrier functions. Diabetic mice treated with exendin-4 showed a significant reduction in all cerebral pathological neovascularisation indices and an improved blood-brain barrier (p<0.05). The vascular protective effects were accompanied by significant improvement in the learning and memory functions of diabetic mice compared with control mice (p<0.05). Our results showed that HBMPCs expressed the GLP-1 receptor. Diabetes increased GLP-1 receptor expression and receptor nitration in HBMPCs. Stimulation of HBMPCs with exendin-4 under diabetic conditions decreased diabetes-induced vascular inflammation and oxidative stress, and restored pericyte function (p<0.05). This study provides novel evidence that brain pericytes express the GLP-1 receptor, which is nitrated under diabetic conditions. GLP-1 receptor activation improves brain pericyte function resulting in restoration of vascular integrity and BBB functions in diabetes. Furthermore, the GLP-1 RA exendin-4 alleviates diabetes-induced cognitive impairment in mice. Restoration of pericyte function in diabetes represents a novel therapeutic target for diabetes-induced cerebrovascular microangiopathy and VCID.

Programmed genomic instability regulates neural transdifferentiation of human brain microvascular pericytes

BackgroundTransdifferentiation describes transformation in vivo of specialized cells from one lineage into another. While there is extensive literature on forced induction of lineage reprogramming in vitro, endogenous mechanisms that govern transdifferentiation remain largely unknown. The observation that human microvascular pericytes transdifferentiate into neurons provided an opportunity to explore the endogenous molecular basis for lineage reprogramming.ResultsWe show that abrupt destabilization of the higher-order chromatin topology that chaperones lineage memory of pericytes is driven by transient global transcriptional arrest. This leads within minutes to localized decompression of the repressed competing higher-order chromatin topology and expression of pro-neural genes. Transition to neural lineage is completed by probabilistic induction of R-loops in key myogenic loci upon re-initiation of RNA polymerase activity, leading to depletion of the myogenic transcriptome and emergence of the neurogenic transcriptome.ConclusionsThese findings suggest that the global transcriptional landscape not only shapes the functional cellular identity of pericytes, but also stabilizes lineage memory by silencing the competing neural program within a repressed chromatin state.

A Human Neurovascular Unit On-a-Chip.

Protection of the central nervous system (CNS) and cerebral homeostasis depend upon the blood-brain barrier (BBB) functions and permeability. BBB restrictive permeability hinders drug delivery for the treatment of several neurodegenerative diseases and brain tumors. Several in vivo animal models and in vitro systems have been developed to understand the BBB complex mechanisms and aid in the design of improved therapeutic strategies. However, there are still many limitations that should be addressed to achieve the structural and chemical environment of a human BBB. We developed a microfluidic-based model of the neurovascular unit. A monolayer of human cerebral endothelial cells (hCMEC-D3) was grown and cocultured with human brain microvascular pericytes (hBMVPC), and human induced pluripotent stem cells differentiated into astrocytes (hiPSC-AC) and neurons (hiPSC-N). To visualize the physiological morphology of each cell type, we used fluorescent cell-specific markers and confocal microscopy. Permeation of fluorescent solutes with different molecular weights was measured to demonstrate that the developed BBB was selectively permeable as a functional barrier.

Abstract P729: Glp-1 Receptor Nitration Contributes to Brain Pericytes Dysfunction in Diabetes

Brain pericytes are unique, multi-functional mural cells located at the center of the NVU. Pericytes maintain the blood-brain barrier (BBB), promote vascular stability, and control the capillary blood flow. We have previously shown that diabetes alters the functions of the pericytes causing BBB damage and increased cerebrovascular pathological neovascularization. GLP-1 agonists, used in the management of diabetes, showed vascular protective effects. However, the underlying mechanisms are unclear. We hypothesis that GLP-1 receptor nitration contributes to the loss of pericytes functions in diabetes. Methods: Expression of GLP-1R in pericytes was assessed in the brain of control and diabetic mice and human brain microvascular pericytes using IHC, RT-PCR, and Western blots. BBB was assessed using Western blot/IHC of Occludin, VEGF, and MMPs gelatinase. Vascular integrity was determined by measuring cerebrovascular neovascularization indices (vascular density, tortuosity, and branching density). GLP-1R nitration was determined by immunoprecipitation and slot blotting. Results: In the present study, we provide novel evidence that the human brain pericytes express the GLP-1R (P&lt;0.05). Moreover, diabetes increased GLP-1R expression in pericytes (P&lt;0.05). Diabetes caused BBB dysfunction, loss of cerebrovascular integrity, and increased all pathological neovascularization indices (P&lt;0.05). Diabetes increased nitration of GLP-1R. Treatment of pericytes with Exendin-4, a GLP-1 agonist, under diabetic conditions, decreased diabetes-induced inflammation, oxidative stress, and pericytes migration, which ameliorated the integrity of the BBB, and prevented the pathological neovascularization (P&lt;0.05). Conclusion: Our results provide novel evidence that pericytes express GLP-1R and that diabetes increased receptor expression and nitration. Treatment of pericytes with GLP-1R agonist exerts neurovascular protective effects, making it a promising strategy in preventing diabetes- mediated cerebrovascular microangiopathy.

Excess adiponectin in eyes with progressive ocular vascular diseases.

Anti-vascular endothelial growth factor (VEGF) therapies are now the first-line treatment for many ocular diseases, but some patients are non-responders to these therapies. The purpose of this study was to determine whether the level of adiponectin increased the pathogenesis of retinal edema and neovascularization in the retina of progressive ocular vascular diseases. We examined the role played by adiponectin in two types of cells and animal models which are retinal vein occlusion (RVO) and oxygen-induced retinopathy (OIR) mice. Our results showed that an injection of anti-adiponectin antibody ameliorated the retinal edema and ischemia through the depression of the expression level of VEGF-related factors and tight junction-related proteins in the retina of RVO mice. The intravitreal injection of anti-adiponectin antibody also decreased the degree of retinal neovascularization in an OIR mice. In addition, exposure of human retinal microvascular endothelial cells and human brain microvascular pericytes in culture to adiponectin increased both the vascular permeability and neovascularization through the increase of inflammatory factor and the dropout of the pericytes. These findings indicate that adiponectin plays a critical role in retinal edema and neovascularization, and adiponectin is a potential therapeutic target for the treatment of diabetic macular edema, proliferative diabetic retinopathy, and RVO.

A subtype of cerebrovascular pericytes is associated with blood-brain barrier disruption that develops during normal aging and simian immunodeficiency virus infection

Lax phenotypic characterization of these morphologically distinct pericytes has delayed our understanding of their role in neurological disorders. We herein establish markers which uniquely distinguish different subpopulations of human brain microvascular pericytes and characterize them independently from cerebrovascular smooth muscle cells. Furthermore, we begin to elucidate the roles of these subsets in blood-brain barrier (BBB) breakdown by studying natural aging and simian immunodeficiency virus (SIV) infection in rhesus macaques. We demonstrate that the main type-1 pericyte subpopulation in the brain of young uninfected adults is positive for platelet-derived growth factor receptor-β (PDGFRB) and negative for α-smooth muscle actin (SMA) and myosin heavy chain 11 (MYH11), whereas PDGFRB+/SMA+/MYH11− (type-2) pericytes are found more frequently in older adults and are associated with SIV infection and progression. Interestingly, we find a strong positive correlation between the degree of BBB breakdown and the percentage of type-2 pericytes regardless of age or SIV status. Taken together, our findings suggest that type-2 pericytes may be a cellular biomarker related to BBB disruption independent of disease status.

Abstract 39: GLP-1 Agonists Improve Cerebrovascular Integrity and Vascular Induced Cognitive Impairment and Dementia Beyond Glycemic Control via Restoration of Brain Pericytes Functions in Diabetic Mice

We have previously shown that diabetes causes pericyte-dysfunction that leads to loss of vascular integrity and vascular-induced cognitive impairment and dementia (VCID). Glucagon-like peptide-1(GLP-1), used in the management of type-2 diabetes mellitus, improves cognitive functions in diabetic patients beyond glycemic control, yet the mechanism is unknown. In the present study, we hypothesis that GLP-1 agonist improves VICD through prevention of diabetes-induced pericytes dysfunction in a non-glucose dependent way. Methods: Control and diabetic mice were randomly assigned for saline or Exendin-4 (GLP-1 agonist 30 ng/kg/day), delivered through osmotic pump over 28 days. Vascular integrity was assessed by measuring cerebrovascular neovascularization indices (Vascular density, tortuosity, and branching density). Cognitive functions were evaluated with Barnes maze and Morrison Water maze. Human brain microvascular pericytes, HBMPCs, were grown in high glucose 25 mM/ sodium palmitate 200 uM (HG/Pal) to mimic diabetic conditions. HBMPCs were treated with/out Exendin-4 and assessed for oxidative stress and angiogenic properties. Results: Diabetic mice treated with GLP-1 agonist showed a significant reduction in all cerebral pathological neovascularization indices (P&lt;0.05). Exendin-4 vascular protective effects were accompanied by significant improvement of the learning and memory functions of diabetic mice (P&lt;0.05). Our results showed that HBMPCs expressed the GLP-1 receptor. Treating HBMPCs exposed to diabetic conditions with GLP-1 agonist restored pericyte functions, decreased diabetes-induced inflammation, oxidative stress, and migration (P&lt;0.05). Conclusion: Our results provide novel evidence that GLP-1 agonist produces neurovascular protective effects in part through targeting pericytes. Restoration of pericyte functions in diabetes represents a novel therapeutic target for diabetes-induced vascular remodeling and VCID.

Inhibition of Ephrin-B2 in brain pericytes decreases cerebral pathological neovascularization in diabetic rats.

We have previously shown that diabetes causes dysfunctional cerebral neovascularization that increases the risk for cerebrovascular disorders such as stroke and cognitive impairment. Pericytes (PCs) play a pivotal role in the angiogenic process through their interaction with the endothelial cells (EC). Yet, the role of PCs in dysfunctional cerebral neovascularization in diabetes is unclear. In the present study, we tested the hypothesis that the increased proangiogenic Ephrin-B2 signaling in PCs contributes to the dysfunctional cerebral neovascularization in diabetes. Type-II diabetes was induced by a combination of high fat diet and low dose streptozotocin injection in male Wistar rats. Selective in vivo Ephrin-B2 silencing in brain PCs was achieved using the stereotactic injection of adeno-associated virus (AAV) with NG2-promoter that expresses Ephrin-B2 shRNA. Neovascularization was assessed using vascular fluorescent dye stain. Novel object recognition (NOR) test was used to determine cognitive functions. Human brain microvascular pericytes HBMVPCs were grown in high glucose 25 mM and palmitate 200 uM (HG/Pal) to mimic diabetic conditions. Scratch migration and tube formation assays were conducted to evaluate PC/EC interaction and angiogenic functions in PC/EC co-culture. Diabetes increased the expression of Ephrin-B2 in the cerebrovasculature and pericytes. Concomitant increases in cerebral neovascularization parameters including vascular density, tortuosity and branching density in diabetic rats were accompanied by deterioration of cognitive function. Inhibition of Ephrin-B2 expression in PCs significantly restored cerebral vascularization and improved cognitive functions. HG/Pal increased PC/EC angiogenic properties in co-culture. Silencing Ephrin-B2 in PCs significantly reduced PC migration and PC/EC co-culture angiogenic properties. This study emphasizes the significant contribution of PCs to the pathological neovascularization in diabetes. Our findings introduce Ephrin-B2 signaling as a promising therapeutic target to improve cerebrovascular integrity in diabetes.

The use of bacterial cellulose as a basement membrane improves the plausibility of the static in vitro blood-brain barrier model

There are several blood-brain barrier (BBB) models available for pharmaceutical research, but none of those are able to properly imitate the permeability of this special barrier. In this study, it is aimed to produce different BBB models with different cellular combinations and different basement membrane polymers, such as polyethylene terephthalate (PET) and bacterial cellulose (BC), which has not been used for BBB models before, to compare their barrier properties. Primary human brain microvascular endothelial cells were seeded on the luminal side and primary human astrocytes and/or primary human brain microvascular pericytes were seeded on the abluminal side of the membranes. Immunofluorescence (IF) staining results indicate that the expression of tight and adherence junction proteins increases on the 5th day of the cultivation. In accordance with Live-Dead staining results, IF images show that cells in the model lose their viability before the 10th day. Transendothelial electrical resistance (TEER) measurements indicate that BC membrane leads to statistically higher (p < 0.05) TEER values than the standard Transwell PET insert membrane. Sucrose and caffeine permeability values of all models are close to in vivo values. BC shows potential to be used as a more reliable basement membrane for BBB models for pharmaceutical research.

Abstract 144: Inhibition of EphrinB2 Expression in Pericytes Decreases Cerebrovascular Pathological Neovascularization in Diabetes

We have previously shown that diabetes causes dysfunctional cerebral neovascularization that increases the risk for cerebrovascular disorders such as stroke and cognitive impairment. Pericytes (PC) play a pivotal role in the angiogenic process through interaction with endothelial cells (EC). Yet, the role of PCs in dysfunctional cerebral neovascularization in diabetes is unclear. In the present study, we tested the hypothesis that the increased proangiogenic EphrinB2 signaling in PCs contributes to the dysfunctional cerebral neovascularization in diabetes. Methods: Type-II diabetes was induced in Wistar rats. Conditional reduction of EphrinB2 expression in rat brain PCs was achieved using stereotactic injection of AAV-virus with NG2-promoter that expresses EphrinB2 shRNA. Neovascularization was assessed using fluorescent space filling model. Novel object recognition (NOR) test was used to assess cognitive functions. Human brain microvascular PCs were grown in glucose 25 mM/palmitate 200 uM (HG/Pal) to mimic diabetic conditions. PC/EC co-culture was used to assess PC/EC interaction and angiogenic functions (scratch migration and tube formation). Results: Diabetes increased expression of EphrinB2 in the cerebrovasculature (2.2-Fold*) and pericytes (1.4 fold*). Diabetes significantly increased cerebral vascularization (Vascular density 3.1 fold*, tortuosity 1.07 fold* and branching density 1.5 fold*) in Wistar rats that were accompanied by deterioration of cognitive function (40% reduction*). Inhibition of EphrinB2 expression in PC significantly restored cerebral vascularization indices back to normal and improved rat cognitive functions with higher D-2 scores in NOR compared to diabetic rats*. HG/Pal increased PC/EC angiogenic properties in coculture (2-fold*). Silencing Ephrin-B2 in HBMVP significantly reduced PC migration (35 %*) and PC/EC co-culture angiogenic properties. (*p&lt;0.05). Conclusion: Our findings emphasize the crucial role that PC plays in the pathological neovascularization in diabetes. EphrinB2 signaling is a promising therapeutic target to improve cerebrovascular integrity in diabetes. Yet, the molecular mechanism of the EphrinB2 signaling in PC/EC interaction is to be uncovered.

Abstract 224: Angiogenic Imbalance Causes Cerebrovascular Pathological Neovascularization in Diabetes: Role of Pericytes.

Diabetes increases cerebrovascular complications such as stroke and cognitive impairment. We have previously showed that diabetes causes dysfunctional cerebral neovascularization. Pericytes have a pivotal role in modulating microvascular physiology and pathology, yet its role in diabetes dysfunctional cerebral neovascularization is unknown. In the present study we testedthe hypothesis that pericytes play a crucial role in diabetes-mediated dysfunctional pathological cerebral neovascularization via dysregulation of antiangiogenic Slit-2/ROBO4 and the proangiogenic EphrinB2/EphB4 signaling. Methods: Diabetes was induced in Wistar rats using low dose of streptozotocin and high fat diet (STZ/HFD). Immunohistochemistry and immunoblotting were used to assess ROBO4 and EphrinB2 expression in the brain and human brain microvascular pericytes (HBMVP). HBMVP was exposed to glucose (25 mM) /palmitate (200 uM) to mimic diabetic conditions. Scratch migration assay was used to assess the effect of high glucose/palmitate (HG/Pal) on pericytes migration. Adenovirus was used to overexpress ROBO4 in the brain in vivo. Results: STZ/HFD caused a significant increase in HbA1C (10.8*). Diabetic brains and HBMVP exposed to HG/Pal showed reduced ROBO4 expression (66%* and 48%*) and increased EphrinB2 expression (1.9* and 1.6* folds). HG/Pal increased pericyte migration by 2* fold. Pericyte migration was increased by 2.5* fold with silencing ROBO4 but significantly reduced with silencing EphrinB2 (35%*) or ROBO4 overexpression (47%*) under HG/Pal. In vivo ROBO4 overexpression decreased pathological neovascularization indices and increased pericyte coverage. (n= 3-5, *P&lt;0.05). Conclusion: Our findings emphasize the importance of balanced anti- and proangiogenic signaling for proper pericyte function in diabetes. Our results suggest that pericytes are involved in proangiogenic and barrier functions of endothelial cells and pericyte dysfunction may contribute to pathological neovascularization in diabetes. Thus, pericyte ROBO4 and EphrinB2 signaling are promising therapeutic targets to improve cerebrovascular integrity in diabetes.

Ascorbic acid efflux from human brain microvascular pericytes: role of re-uptake.

Microvascular pericytes take up ascorbic acid on the ascorbate transporter SVCT2. Intracellular ascorbate then protects the cells against apoptosis induced by culture at diabetic glucose concentrations. To investigate whether pericytes might also provide ascorbate to the underlying endothelial cells, we studied ascorbate efflux from human pericytes. When loaded with ascorbate to intracellular concentrations of 0.8-1.0 mM, almost two-thirds of intracellular ascorbate effluxed from the cells over 2 H. This efflux was opposed by ascorbate re-uptake from the medium, since preventing re-uptake by destroying extracellular ascorbate with ascorbate oxidase increased ascorbate loss even further. Ascorbate re-uptake occurred on the SVCT2, since its blockade by replacing medium sodium with choline, by the SVCT2 inhibitor sulfinpyrazone, or by extracellular ascorbate accelerated ascorbate loss from the cells. This was supported by finding that net efflux of radiolabeled ascorbate was increased by unlabeled extracellular ascorbate with a half-maximal effect in the range of the high affinity Km of the SVCT2. Intracellular ascorbate did not inhibit its efflux. To assess the mechanism of ascorbate efflux, known inhibitors of volume-regulated anion channels (VRACs) were tested. These potently inhibited ascorbate transport into cells on the SVCT2, but not its efflux. An exception was the anion transport inhibitor DIDS, which, despite inhibition of ascorbate uptake, also inhibited net efflux at 25-50 µM. These results suggest that ascorbate efflux from vascular pericytes occurs on a DIDS-inhibitable transporter or channel different from VRACs. Further, ascorbate efflux is opposed by re-uptake of ascorbate on the SVCT2, providing a potential regulatory mechanism.

Pericytes Contribute to the Disruption of the Cerebral Endothelial Barrier via Increasing VEGF Expression: Implications for Stroke

Disruption of the blood-brain barrier (BBB) integrity occurring during the early onset of stroke is not only a consequence of, but also contributes to the further progression of stroke. Although it has been well documented that brain microvascular endothelial cells and astrocytes play a critical role in the maintenance of BBB integrity, pericytes, sandwiched between endothelial cells and astrocytes, remain poorly studied in the pathogenesis of stroke. Our findings demonstrated that treatment of human brain microvascular pericytes with sodium cyanide (NaCN) and glucose deprivation resulted in increased expression of vascular endothelial growth factor (VEGF) via the activation of tyrosine kinase Src, with downstream activation of mitogen activated protein kinase and PI3K/Akt pathways and subsequent translocation of NF-κB into the nucleus. Conditioned medium from NaCN-treated pericytes led to increased permeability of endothelial cells, and this effect was significantly inhibited by VEGF-neutralizing antibody. The in vivo relevance of these findings was further corroborated in the stroke model of mice wherein the mice, demonstrated disruption of the BBB integrity and concomitant increase in the expression of VEGF in the brain tissue as well as in the isolated microvessel. These findings thus suggest the role of pericyte-derived VEGF in modulating increased permeability of BBB during stroke. Understanding the regulation of VEGF expression could open new avenues for the development of potential therapeutic targets for stroke and other neurological disease.

The effect of AMA0428, a novel and potent ROCK inhibitor, in a model of neovascular age-related macular degeneration.

Rho kinase (ROCK) is associated with VEGF-driven angiogenesis, as well as with inflammation and fibrosis. Therefore, the effect of AMA0428, a novel ROCK inhibitor, was studied in these processes, which highly contribute to the pathogenesis of neovascular AMD. The effect of AMA0428 (0.5-5.0 μM) on human umbilical vein endothelial cells (HUVECs), human brain microvascular endothelial cells (HBMECs), and human brain microvascular pericytes (HBVPs) was determined using cell viability (WST-1), apoptosis (caspase 3/7), and migration (scratch and under-agarose) assays. The in vivo response was investigated using a laser-induced choroidal neovascularization (CNV) mouse model, in which intravitreal injections of AMA0428, murine anti-VEGF-R2 mAb (DC101), or placebo was given. Outcome was assessed by analysis of inflammation (CD45), angiogenesis (FITC-dextran), vessel leakage (Texas Red-conjugated Dextran and FITC-labeled lectin) and fibrosis (Sirius Red/Collagen I). The AMA0428 dose-dependently reduced proliferation and VEGF-induced migration of HUVEC and HBMEC (P < 0.05). No significant effect was seen on HBVP proliferation; however, migration and pericyte recruitment were enhanced (P < 0.05) by AMA0428 administration. There was no apoptosis induction. The AMA0428 significantly reduced CNV and vessel leakage 2 weeks after laser treatment, comparable to DC101. In addition, AMA0428 inhibited inflammation on day 5 by 42% (P < 0.05) and collagen deposition on day 30 by 43% (P < 0. 05), whereas DC101 had no effect on inflammation nor on fibrosis. The results suggest that targeting ROCK with AMA0428 not only reduces neoangiogenesis, but also blocks inflammation and fibrosis (contrary to VEGF suppression). These results point to a potential therapeutic benefit of ROCK inhibition in neovascular AMD.

Tat 101-mediated enhancement of brain pericyte migration involves platelet-derived growth factor subunit B homodimer: implications for human immunodeficiency virus-associated neurocognitive disorders.

In the era of antiretroviral therapy, although the human immunodeficiency virus (HIV) replication can be successfully controlled, complications of the CNS continue to affect infected individuals. Viral Tat protein is not only neurotoxic but has also been shown to disrupt the integrity of the blood-brain barrier (BBB). Although the role of brain microvascular endothelial cells and astrocytes in Tat-mediated impairment has been well documented, pericytes, which are important constituents of the BBB and play a key role in maintaining the integrity of the barrier, remain poorly studied in the context of HIV-associated neurocognitive disorders (HAND). In the present study, we demonstrated that exposure of human brain microvascular pericytes and C3H/10T1/2 cells to HIV-1 Tat101 resulted in increased expression of platelet-derived growth factor subunit B homodimer (PDGF-BB) and increased migration of the treated cells. Furthermore, we also demonstrated that this effect of Tat was mediated via activation of mitogen-activated protein kinases and nuclear factor-κB pathways. Secreted PDGF-BB resulted in autocrine activation of the PDGF-BB/PDGF β receptor signaling pathway, culminating ultimately into increased pericyte migration. Ex vivo relevance of these findings was further corroborated in isolated microvessels of HIV Tg26 mice that demonstrated significantly increased expression of PDGF-BB in isolated brain microvessels with a concomitant loss of pericytes. Intriguingly, loss of pericyte coverage was also detected in sections of frontal cortex from humans with HIV-encephalitis compared with the uninfected controls. These findings thus implicate a novel role of PDGF-BB in the migration of pericytes, resulting in loss of pericyte coverage from the endothelium with a subsequent breach of the BBB.

Induction of aldose reductase expression and reduction of sorbitol dehydrogenase expression by methylmercury in cultured human brain microvascular pericytes

Induction of aldose reductase expression and reduction of sorbitol dehydrogenase expression by methylmercury in cultured human brain microvascular pericytes

Cell-density-dependent methylmercury susceptibility of cultured human brain microvascular pericytes

The knowledge of vascular toxicity is important for understanding the neurotoxicity of methylmercury. In the present study, we investigated the cell-density-dependent susceptibility of human brain microvascular pericytes to methylmercury-induced toxicity by using a cell-culture system. The susceptibility of sparse pericyte cultures to methylmercury was greater than that of the dense cultures. In addition, the sparse cultures were more susceptible to methylmercury than to inorganic mercury and cadmium. The intracellular accumulation of methylmercury in the sparse cells was significantly higher than that in the dense cells. Methylmercury is transported through the L-type large neutral amino acid transporter (LAT 1) in the form of a complex with cysteine. The mRNA- and protein-level expressions of LAT 1 in the sparse cells were markedly higher than those in the dense cells; in addition, the LAT 1 expression was increased by methylmercury. However, there was no reduction in the levels of glutathione and metallothionein, which are involved in the defense mechanisms against methylmercury, in the sparse cells. The present data revealed that pericytes are markedly susceptible to methylmercury-induced cytotoxicity at low cell densities. The susceptibility of the sparse pericytes is postulated to be due to the not only constitutively higher but also methylmercury-induced expression of LAT 1, which increased the intracellular accumulation of methylmercury.

Cilostazol Induces Metallothionein Expression in Vascular Cells

Metallothionein (MT) is a cysteine-rich low molecular weight protein and thought to function in the detoxification of heavy metals and reactive oxygen species. We examined the induction of MT synthesis by cilostazol, an antiplatelet drug, in several vascular component cells to find a new pharmacological effect of cilostazol in vascular system. In human coronary artery endothelial cells, cilostazol significantly increased MT-IX and MT-IIA mRNA levels after the treatment for 6 h and endogenous MT-I/II protein levels after the treatment for 24 and 48 h. In addition, cadmium cytotoxicity was prevented by cilostazol in this endothelial cells. Moreover, cilostazol increased MT-IX and MT-IIA mRNA levels in human coronary artery smooth muscle cells, human brain microvascular endothelial cells, human brain microvascular pericytes and human colon carcinoma Caco-2 cells after the treatment for 6 h. Interestingly, cilostazol also increased MT-III mRNA level in brain microvascular endothelial cells more effectively than in other vascular cells. The present results suggest that cilostazol can protect the vascular system from toxic substances such as heavy metals via MT induction in vascular cells.