The mechanism of the receptors for advanced glycation end products affecting pericyte detachment from blood vessels after traumatic brain injury / 西安交通大学学报(医学版)

【Objective】 To observe the reactive change of cortical perivascular cells after craniocerebral injury and explore its mechanism. 【Methods】 The controllable cortical impact animal model was used to simulate craniocerebral injury, the expressions of cortical pericyte markers at different time points after trauma were studied by Western blotting, and the biological behavior of vascular pericytes after craniocerebral injury was determined by transmission electron microscopy. Post-traumatic high mobility group box 1 (HMGB1), receptor for advanced glycation end product (RAGE), and nuclear factor κB (NF-κB) were detected by Western blotting. The experimental animals were divided into FPS-ZM1 (a specific RAGE receptor blocker) injection group and wild-type group. Wet and dry brain weight and transmission electron microscopy were used to study the post-traumatic effects of HMGB1-RAGE on pericytes. The primary mouse brain microvascular pericytes were cultured and supplemented with HMGB1 recombinant protein; the cultured pericytes supplemented with FPS-ZM1 were used as the control to explore the effect of HMGB1-RAGE pathway on vascular pericytes in vitro. 【Results】 The expression levels of early post-traumatic cortical pericyte markers platelet-derived growth factor receptor beta (PDGFR-β) and NG2 proteoglycan (NG2) decreased (PDGFR-β, Control vs. CCI 3D P<0.05; NG2, Control vs. CCI 6H P<0.05; Control vs. CCI 1D P<0.05). We found that pericytes were detached from blood vessels, accompanied by local blood-brain barrier opening. The expression of HMGB1-RAGE-NF-κB signaling pathway was increased in the early cortex after trauma (HMGB1, Control vs. CCI 6H P<0.05, Control vs. CCI 1D P<0.05; RAGE, Control vs. CCI 6H P<0.05, Control vs. CCI 1D P<0.05, Control vs. CCI 3D P<0.05, Control vs. CCI 5D P<0.05, Control vs. CCI 7D P<0.05; NF-κB, Control vs. CCI 6H P<0.05, Control vs. CCI 1D P<0.05). After blocking the binding of RAGE with the ligand, cortical edema was reduced (CCI 6H P<0.05, CCI 1D P<0.05), and neurovascular unit damage was reduced. HMGB1 recombinant protein could increase the migration ability of cultured pericytes (Control vs. HMGB1 P<0.05, Control vs. HMGB1+FPS-ZM1 P<0.05), and could be reversed by FPS-ZM1 (HMGB1 vs. HMGB1+FPS-ZM1 P<0.05). 【Conclusion】 High-level HMGB1 after traumatic brain injury mediates pericytes’ detachment from blood vessels through RAGE on pericytes and leads to the occurrence of local cerebral edema.

Correlation between cerebrospinal fluid soluble platelet-derived growth factor receptor β and cognitive damage and cerebrospinal fluid core biological markers in Alzheimer′s disease / 中华神经科杂志

Objective:To investigate the relationship between the concentration of soluble platelet-derived growth factor receptor β (sPDGFRβ) in the cerebrospinal fluid (CSF) of patients with Alzheimer′s disease (AD) and the degree of cognitive impairment and cerebrospinal fluid biomarkers.Methods:A total of 50 patients with AD in the Department of Neurology of Provincial Hospital Affiliated to Anhui Medical University from September 2018 to August 2020 were selected as AD group, and 33 patients with normal cognition who had no significant difference in age and gender in the same period served as control group. The neuropsychological evaluation was conducted. According to the Clinical Dementia Rating scale scores, the AD patients were divided into mild AD group and moderate to severe AD group.The clinical data and cognitive function of the three groups were compared. And the level of CSF sPDGFRβ, CSF amyloid-β (Aβ) 1-40, CSF Aβ 1-42, CSF total tau protein (T-tau), CSF phosphorylated tau protein (P-tau) were measured by enzyme linked immunosorbent assay in each group. According to whether apolipoprotein E4 (ApoE4) gene was carried, the patients with AD were divided into ApoE4 + group and ApoE4 - group. Differences among the three groups were compared and the correlation analysis was carried out. Results:The levels of sPDGFRβ in the CSF of the mild AD group [(219.301±69.711) pg/ml] and the moderate to severe AD group [(235.358±86.187) pg/ml] were significantly higher than that of the control group [(184.878±52.944) pg/ml, F=3.90, P=0.024], while there was no significant difference in the level of CSF sPDGFRβ between the ApoE4 + group [(219.493±76.745) pg/ml] and the ApoE4 - group [(222.802±81.665) pg/ml, t=-0.13, P=0.900]. And the level of sPDGFRβ in the CSF in the mild AD group was positively correlated with the level of CSF P-tau ( r=0.43, P=0.019), but not correlated with Aβ 1-42, T-tau, Mini-Mental State Examination scores or Montreal Cognitive Assessment scores, whereas no significant correlation was found in the control group and the moderate to severe AD group. Conclusions:Expression of sPDGFRβ in CSF of AD patients is increased, and may relate to P-tau. Pericyte injury may be involved in the phosphorylation of tau protein in the brain of AD patients.

Progress of pulmonary pericytes in lung development and lung diseases / 国际儿科学杂志

As a member of vessel extracellular matrix, pericytes have a close relationship with angiogenesis.Pericytes widely exist on the surface of microvascular all over the body, which are important to the stability of vessels.As a member in development of vascular structure, pericytes are located in the niches between the junction of lung vascular cells, and pericytes definitely play important roles in lung development and lung diseases.Based on the functional characters of pericytes, this article summarizes the relevant researches about the roles of pericytes in the lung development and lung diseases, and further study of pericytes will provide new insights into the mechanism of lung development and lung diseases.

The activation of hippocampal pericytes and glial scar formation in rats with status epilepticus / 国际儿科学杂志

Objective:To explore the perivascular activation of reactive pericytes after status epilepticus(SE), and the relationship between pericytes and glial cells in proliferation and function.Methods:Eighty rats were randomly divided into control group( n=16) and model group( n=64, 16 for each group in SE1d, SE3d, SE7d, SE28d). The SE model was induced by intraperitoneal injection of lithium chloride and pilocarpine, and hematoxylin-eosin staining was performed on brain tissue sections to observe basic pathological changes.Use immunohistochemistry and Western blot to detect(neuron-glial antigen 2, NG2) expression, and use immunofluorescence technology to double stain NG2 and(glial fibrillary acidic protein, GFAP) to observe their relationship. Results:In the model group, the neurons were arranged disorderly, losing the ribbon structure, and the neurons appeared degeneration and necrosis.It was observed that the nuclei of the neurons were blurred, and the cytoplasm was agglomerated.There were more glial cells proliferation.Compared with the control group, it was found in model group that NG2 showed a dynamic high expression after SE( P<0.05). The number of pericytes increased significantly, reaching a peak at 7d, and the results of Western blot were consistent with the results of histochemistry( P<0.05). The aggregation of glial cells were induced in the surrounding area, and pericytes participated in the signal transduction of glial cells. Conclusion:Pericytes can induce the aggregation of glial cells and participate in the repairment in the form of glial scars after SE brain injury.

Changes of pdgfr-β+ microvascular wall cells in blood-brain barrier permeability of SD rats after status epilepticus / 国际儿科学杂志

Objective To investigate the blood-brain barrier permeability of platelet-derived growth factor receptor-β + (pdgfi-β +) vascular wall cells at 2h,24h,3 d,7 d,14 d and 21 d after lithium-pilocarpine-inducedstatus epilepticus.Methods One hundred and thirty-five clean male Sprague-Dawley rats were randomly divided into control group (n =15) and model group (n =120).The model group was divided into 2h,24h,3d,7d,14d and21d after SE.We evaluated the permeability of blood-brain barrier in hippocampus of rats in each group after status epilepticus by Evans blue method and wet and dry weight method.We observed the ultrastructural changes of pericytes and blood-brain barriers at different stages after onset by electron microscopy.We used Western Blot to detect the expression of pericyte marker pdgfr-β and α-SMA in hippocampus at different stages after onset.Results (1) The blood-brain barrier permeability increased after epileptic seizures (P ＜ 0.05),and the permeability was the highest at 24h after onset (P ＜ 0.01),and gradually returned to normal after 3d and later.(2) Transmission electron microscopy showed that the ultrastructure of cerebral microvascular pericytes and their basement membranes were degenerated after SE.(3) Western blot showed that the expression level of pdgfr-β and α-SMA at 24 h after SE was significantly higher than that of the control group (P ＜0.05),and gradually became stable after 3d and later.Conclusion Pdgfr-β + microvascular wall cells in brain microvessels may be involved in the opening of blood-brain barrier after status epilepticus,and may be dedicated to the conversion of disease into refractory epilepsy.

Pericyte loss and detachment in experimental cisplatin-induced neuropathy / Pérdida y desprendimiento de pericitos en la neuropatía experimental inducida por cisplatino

Cisplatin is an antineoplastic agent with neuropathy as one of its major side effect. However, effective treatment is lacking. Increasing evidence suggests that cisplatin might damage nerve capillaries leading to impaired functions of blood-nerve barrier (BNB) and neuropathy. This study was aimed to examine the effects of cisplatin on pericytes. Rats were either treated with intraperitoneal injection of cisplatin 2 mg/kg twice a week for five continuous weeks. Cisplatin-treated rats showed reduced body weight, thermal hypoalgesia and slow sciatic motor nerve conduction velocity, indicating neuropathy. The density of pericytes in the distal sciatic nerves determined by immunohistochemistry to desmin was significantly reduced in the cisplatin compared with that of the control groups. Electron microscopic analysis demonstrated the detachment of pericytes from endothelial cells including the disruption of shared basement membrane in the sciatic nerves from cisplatin-treated rats. These data indicate the pericyte loss and detachment caused by cisplatin. Future studies of the BNB components and functions after cisplatin treatment are needed and will be essential for the development of effective treatments against cisplatin-induced neuropathy.

El cisplatino es un agente antineoplásico y presenta como uno de sus principales efectos secundarios, la neuropatía. Sin embargo, falta un tratamiento eficaz. La creciente evidencia sugiere que el cisplatino podría dañar los capilares nerviosos, lo que puede provocar una alteración de las funciones de la barrera hematoencefálica (BHE) y neuropatía. Este estudio tuvo como objetivo examinar los efectos del cisplatino en los pericitos. Las ratas se trataron con inyección intraperitoneal de cisplatino (2 mg/kg) dos veces por semana durante 5 semanas seguidas. Las ratas tratadas con cisplatino mostraron una reducción del peso corporal, hipoalgesia térmica y una velocidad de conducción del nervio ciático lenta, lo que indicaría neuropatía. La densidad de los pericitos en los nervios ciáticos distales determinada por inmunohistoquímica para desmina se redujo significativamente en el grupo cisplatino en comparación con la de los grupos controles. El análisis al microscopio electrónico demostró el desprendimiento de pericitos de las células endoteliales, incluida la ruptura de la membrana basal compartida en los nervios ciáticos de ratas tratadas con cisplatino. Estos datos indican la pérdida de pericitos y el desprendimiento causado por el cisplatino. Se necesitan estudios futuros de los componentes y funciones del BHE después del tratamiento con cisplatino y serán esenciales para el desarrollo de tratamientos efectivos contra la neuropatía inducida por el cisplatino.

Proteomic research of human retinal microvascular pericytes stimulated with high glucose / 中华实验眼科杂志

Objective@#To determine the changes of protein expressions in human retinal microvascular pericytes (HRMPCs) stimulated with high glucose by using quantitative proteomics, which provides new clues for future investigation of diabetic retinopathy (DR).@*Methods@#HRMPCs were divided into two groups.The cells in control group were cultured in DMEM basic medium with 25 mmol/L glucose, while the cells in high glucose group were cultured in DMEM medium with 35 mmol/L glucose.The amount of living cells was measured by cell counting kit-8(CCK-8). The proteins were collected from the two groups and then were digested with trypsin.Peptides of 2 μg were injected into the time of flight-mass spectrometer and the acquisition mode was DDA.The results were further analyzed using bioinformatics software.@*Results@#CCK-8 results showed that the absorbance (A450) of HRMPCs in high glucose group was 0.75±0.04, which was significantly lower than 0.91±0.05 in control group (t=5.784, P=0.000 2). In total, 1 972 proteins were identified and 54 of them were significantly different between the two groups (fold change >1.5). Among them, 13 proteins were up-regulated, including CTNNB1 and CTBP2; while 41 proteins were down-regulated, including SQSTM1 and HMGCS1.The differentially expressed proteins were mainly involved in citric acid cycle and aerobic respiration.@*Conclusions@#The expressions of many proteins in HRMPCs change under the stimulation of high glucose, which may influence the respiration and the ATP production of cells and eventually induce the loss of pericyte.

Purification and cultivation of mouse primary retinal microvascular pericytes based on pre-incubation / 中华实验眼科杂志

Objective@#To establish a simple method for isolation, purification and cultivation of primary retinal microvascular pericytes (RMPs) from mice.@*Methods@#Retinas were isolated from mice following with mechanical morcel, enzymatic digestion and filtration.The retinal fragments were incubated with low glucose DMEM with 20% fetal bovine serum after 24 hours pre-incubation.Differential digestion was used for purification of primary RMPs.Morphological examination of cells was performed by phase contrast microscopy, and further characterization was analyzed by immunocytochemistry.Functional assay was evaluated by the pericytes-endothelial cells (ECs) co-culture system.The treatment and use of experimental animals followed the regulations on the administration of experimental animals promulgated by the state science and technology commission.@*Results@#Cells migrated out of fragments after 24 hours of incubation, and developed into small or large colonies gradually.The cells and their subpassages presented typical pericyte morphology with large irregular triangular cell bodies and multiple long processes.No contact inhibition was observed.Most cells uniformly expressed the cellular markers α-smooth muscle actin (α-SMA) and platelet-derived growth factor receptor-β (PDGFR-β), a few cells expressed the cellular markers glial fibrillary acidic protein (GFAP), but no cell expressed von Willebrand factor (vWF). The purity rate of RMPs was up to 97%.In the co-culture system, RMPs directly contacted with ECs to form the capillary-like cords in vitro.@*Conclusions@#A simple method for the isolation, purification cultivation of mouse RMPs is established, and active RMPs can be readily obtained by this method.

Purification and cultivation of mouse primary retinal microvascular pericytes based on pre-incubation / 中华实验眼科杂志

Objective To establish a simple method for isolation, purification and cultivation of primary retinal microvascular pericytes ( RMPs) from mice. Methods Retinas were isolated from mice following with mechanical morcel,enzymatic digestion and filtration. The retinal fragments were incubated with low glucose DMEM with 20％ fetal bovine serum after 24 hours pre-incubation. Differential digestion was used for purification of primary RMPs. Morphological examination of cells was performed by phase contrast microscopy, and further characterization was analyzed by immunocytochemistry. Functional assay was evaluated by the pericytes-endothelial cells ( ECs) co-culture system. The treatment and use of experimental animals followed the regulations on the administration of experimental animals promulgated by the state science and technology commission. Results Cells migrated out of fragments after 24 hours of incubation, and developed into small or large colonies gradually. The cells and their subpassages presented typical pericyte morphology with large irregular triangular cell bodies and multiple long processes. No contact inhibition was observed. Most cells uniformly expressed the cellular markers α-smooth muscle actin (α-SMA) and platelet-derived growth factor receptor-β( PDGFR-β) ,a few cells expressed the cellular markers glial fibrillary acidic protein ( GFAP) ,but no cell expressed von Willebrand factor ( vWF) . The purity rate of RMPs was up to 97％. In the co-culture system,RMPs directly contacted with ECs to form the capillary-like cords in vitro. Conclusions A simple method for the isolation, purification cultivation of mouse RMPs is established, and active RMPs can be readily obtained by this method.

Proteomic research of human retinal microvascular pericytes stimulated with high glucose / 中华实验眼科杂志

Objective To determine the changes of protein expressions in human retinal microvascular pericytes ( HRMPCs) stimulated with high glucose by using quantitative proteomics, which provides new clues for future investigation of diabetic retinopathy ( DR) . Methods HRMPCs were divided into two groups. The cells in control group were cultured in DMEM basic medium with 25 mmol/L glucose,while the cells in high glucose group were cultured in DMEM medium with 35 mmol/L glucose. The amount of living cells was measured by cell counting kit-8(CCK-8). The proteins were collected from the two groups and then were digested with trypsin. Peptides of 2μg were injected into the time of flight-mass spectrometer and the acquisition mode was DDA. The results were further analyzed using bioinformatics software. Results CCK-8 results showed that the absorbance ( A450 ) of HRMPCs in high glucose group was 0. 75±0. 04,which was significantly lower than 0. 91±0. 05 in control group (t=5. 784,P=0. 0002). In total,1972 proteins were identified and 54 of them were significantly different between the two groups (fold change >1. 5). Among them,13 proteins were up-regulated,including CTNNB1 and CTBP2;while 41 proteins were down-regulated,including SQSTM1 and HMGCS1. The differentially expressed proteins were mainly involved in citric acid cycle and aerobic respiration. Conclusions The expressions of many proteins in HRMPCs change under the stimulation of high glucose,which may influence the respiration and the ATP production of cells and eventually induce the loss of pericyte.

Pathomechanisms of pericyte-myofibroblast transition in kidney and interventional effects of Chinese herbal medicine / 中国中药杂志

In the kidney, pericyte is the major source of myofibroblast (MyoF) in renal interstitium. It is reported that pericyte-myofibroblast transition（PMT）is one of the important pathomechanisms of renal interstitial fibrosis（RIF）. Among them, the main reasons for promoting RIF formation include pericyte recruitment, activation and isolation, as well as the lack of pericyte-derived erythropoietin. During the PMT startup process, pericyte activation and its separation from microvessels are controlled by multiple signal transduction pathways, such as transforming growth factor-β（TGF-β）pathway, vascular endothelial growth factor receptor (VEGFR) pathway and platelet derived growth factor receptor (PDGFR) pathway;Blocking of these signaling pathways can not only inhibit PMT, but also suppress renal capillaries reduction and further alleviate RIF. In clinic, many traditional Chinese medicine compound prescriptions, single traditional Chinese herbal medicine (CHM) and their extracts have the clear effects in alleviating RIF, and some of their intervention actions may be related to pericyte and its PMT. Therefore, the studies on PMT and its drug intervention will become the main development direction in the research field of anti-organ fibrosis by CHM.

Effects of basic fibroblast growth factor on pericytes in mice with traumatic brain injury / 中华创伤杂志

Objective To investigate the effects of basic fibroblast growth factor (bFGF) on pericytes in the blood brain barrier at acute stage after traumatic brain injury (TBI) in mice.Methods A total of 90 mice with a C57BL/6 background were randomly divided into sham group,TBI group,and TBI + bFGF group,with 30 rats per group.The models of moderate TBl were established using the controlled cortical impactor.After 24 hours,the changes of nerve function were evaluated by Garcia neurological score.Each mouse received an intraperitoneal injection of Evans blue dye for measuring the permeability of blood brain barrier.Western blot was used to test the related indices of pericytes after the cerebral cortex was quickly dissected:platelet-derived growth factor receptor beta (PDGFR-β),aminopeptidase N (CD13),desmin,neurogliocyte 2 (NG2),and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).Paraffin sections were prepared for HE staining and morphological changes were observed.Immunofluorescence assay was used to test the related indices of pericytes:PDGFR-β,CD13,and cell surface glycoprotein MUC18 (CD146).Results Garcia neurological score revealed that the score in TBI group was significantly decreased compared with that in sham group (P ＜ 0.01),but the score of TBI + bFGF group was significantly increased compared with that of TBI group (P ＜ 0.05).Permeability of blood brain barrier in TBI group was significantly increased compared with that in sham group (P ＜0.01),but in TBI + bFGF group this parameter significantly reduced compared with that in TBI group (P ＜ 0.01).Western blot analysis revealed that the expressions of PDGFR-β,CD13,desmin,NG2 proteins in TBI group were significantly decreased compared with those in sham group (P ＜0.05),while the expressions of PDGFR-β,CD13,desmin,NG2 proteins in TBI + bFGF group were significantly increased compared with those in TBI group (P ＜ 0.05).HE staining revealed injury of brain parenchyma in TBI group was the severest compared with both sham group and TBI + bFGF group.Immunofluorescence staining results revealed that the proteins expressions of PDGFR-β,CD13,and CD146 in TBI group were significantly decreased compared with those in sham group (all P ＜0.01),and those in TBI + bFGF group were significantly increased compared with those in TBI group (all P ＜ 0.05).Conclusions bFGF can prevent pericyte death via protecting its proteins to conserve blood-brain barrier,bFGF can also significantly ameliorate the injury of brain parenchyma.

Advances in Shh signaling pathway of renal interstitial fibrosis / 国际儿科学杂志

Sonic hedgehog signaling pathway has a unique role in the process of renal fibrosis,while it is mainly expressed in the impaired renal tubular epithelial cells. Shh signaling pathway promotes the process of epithelial-mesenchymal transition and renal tubulointerstitial fibrosis. Shh signaling pathway ligand triggers cell proliferation in the pericyte,suggesting a possible role for this pathway in the regulation of cell cycle progression of myofibroblasts progenitors during the development of renal fibrosis. Shh protein can be expressed at the early stage of kidney injury,which indicates that Shh signaling pathway is involved in the tissue repair process after kidney injury at the early stage. The process of renal interstitial fibrosis related to signaling pathway is involved with signaling pathway and proteins,cytokines,extracellular matrix,complement and so on. This paper reviews the progress in the study of the relationship between Shh and renal interstitial fibrosis.

Progresses of pericytes in central nervous system microvascular diseases / 国际儿科学杂志

Pericyte is a type of important cellular constituents in the central nervous system (CNS),which is located at periphery of the microvessel wall,surrounded by basement membrane (BM) and connected with endothelial cell (EC) and astrocytes endfeet.As such important components of microvasculature,diverse functions have been assigned to pericytes,including control of brain blood flow,development and maintenance of the blood-brain barrier,regulation of the neuroinflammation and may function as pluripotent stem cells.The dysfunction of brain pericytes is associated with the progression of many CNS diseases.The occurrence of the disease leads to pericytes injury.The dysfunction of the brain pericytes make the disease worse.Increasing evidence suggests a highly specialized role for pericytes in a wide range of CNS diseases like Alzheimer's disease,stroke,subarachnoid hemorrhage,epilepsy and so on.

Study on the effect of single dose intraosseous injection of simvastatin on tumor vascular normalization in murine breast cancer / 中国比较医学杂志

Objective To investigate the effect of single dose intraosseous injection of simvastatin on tumor vascular structure and function in murine breast cancer. Methods BALB/c mice and 4T1 murine breast cancer cells were used to establish a subcutaneous xenograft model. The mouse model of orthotopic breast cancer received intraosseous injection of a single dose of simvastatin (50 μg) or vehicle only. Frozen tumor tissue sections were prepared for co-immunostained with CD31 andα-SMA. Evans blue dye was injected into the tail vein to observe the vascular permeability. The expression level of HIF-1αwas detected by immunohistochemistry. Results Immunofluorescence dual staining showed that intraosseous injection of simvastatin increased the number of perivascular pericytes in the tumor vessel(P < 0. 05), Evans blue dye content showed that in vivo vessel permeability in the tumor tissue was significantly decreased(P < 0. 05), and the immunohistochemistry results showed that local hypoxic area was significantly improved. Conclusions Single dose intraosseous injection of simvastatin can promote the normalization of tumor vasculature by improving the coverage of pericytes.

Study on the effect of single dose intraosseous injection of simvastatin on tumor vascular normalization in murine breast cancer / 中国比较医学杂志

Objective To investigate the effect of single dose intraosseous injection of simvastatin on tumor vascular structure and function in murine breast cancer. Methods BALB/c mice and 4T1 murine breast cancer cells were used to establish a subcutaneous xenograft model. The mouse model of orthotopic breast cancer received intraosseous injection of a single dose of simvastatin (50 μg) or vehicle only. Frozen tumor tissue sections were prepared for co-immunostained with CD31 andα-SMA. Evans blue dye was injected into the tail vein to observe the vascular permeability. The expression level of HIF-1αwas detected by immunohistochemistry. Results Immunofluorescence dual staining showed that intraosseous injection of simvastatin increased the number of perivascular pericytes in the tumor vessel(P < 0. 05), Evans blue dye content showed that in vivo vessel permeability in the tumor tissue was significantly decreased(P < 0. 05), and the immunohistochemistry results showed that local hypoxic area was significantly improved. Conclusions Single dose intraosseous injection of simvastatin can promote the normalization of tumor vasculature by improving the coverage of pericytes.

Expression of Nestin on Endothelial Cells and Pericytes During Retinal Vascular Development in Mouse

PURPOSE: Nestin, a marker of neural stem cells, is expressed in Müller cells during retinal development. However, the role of nestin in retinal vascular development is not well established. Thus, we investigated the expression of nestin in developmental mouse retina and identified which retinal cells are related to the expression of nestin during the retinal vascular development. METHODS: Eyes were enucleated from C57BL/6 mice on postnatal day (P) 4, P8, P12, P16 and P26. Immunofluorescence was used to evaluate nestin expression in relation to endothelial cells (isolectin B4), pericytes (neural/glial antigen 2) and astrocytes (glial fibrillary acidic protein). RESULTS: Nestin was strongly expressed from the ganglion cell layer to retinoblast layer at P4. At P8, P12 and P16, the expression of nestin was observed from the upper border of the ganglion cell layer, and vertically penetrating to outer nuclear layer. At P26, the expression of nestin was decreased and confined to the ganglion cell layer and inner nuclear layer. Interestingly, there was strong vascular shape expression of nestin at all stages. The superficial, deep and intermediate vascular plexus was completely merged with nestin expression at P4, P8, P12 and P16. In addition, the nestin expression merged with pericytes but not with astrocytes. CONCLUSIONS: Nestin was expressed in endothelial cells and pericytes during retinal vascular development in the retina. These results suggest that nestin could play an important role in developmental angiogenesis via interplay with endothelial cells and pericytes.

Angiomyomatous hamartoma in an inguinal lymph node with proliferating pericytes/smooth muscle cells, plexiform vessel tangles, and ectopic calcification.

Angiomyomatous hamartoma (AMH) is a rare benign lesion, which occurs almost exclusively in the inguinal lymph nodes. We herein report a case of a female elder who presented with a long-standing inguinal mass. Microscopically, the mass showed a zonal distribution, characterized by thick-walled muscular vessels, fi brosis, and calcifi cation in the hilum and proliferating spindle cells around thin-walled vessels and plexiform vessel tangles at the periphery. The spindle cells show positive immunoreactivity of smooth muscle actin and CD34 with a heterogeneous expression of desmin and CD44. Although the pathogenesis of AMH remains uncertain, the histological features and immunohistochemical fi ndings of this case imply a disordered or exuberant angiogenic process.

Selective culture of rat retinal microvascular pericytes / 中华实验眼科杂志

Background Retinal microvascular pericytes (RMPs) have been played increasing attention as an emerging key in pathogenesis of various retinal angiogenic diseases including diabetic retinopathy,and RMPs are thought to be a potential target for treatment.Yet the study has been hindered by the difficulty of obtaining source of tissue and isolating pure population.Objective This study was to establish a simple method of isolation,purification and cultivation of primary RMPs for rat.Methods Eyeballs were extracted from clean male Sprague Dawley rats and immersed by 75％ alcohol for 1 minute.The retinas were isolated and mechanical morcel.Trypsin (2.5 g/L) was firstly used and followed by type Ⅰ collagenase (2 g/L) for the digestion of the retina for 15 minutes,respectively.Retinal microvascular fragments were screened by 100 μm and 55 μm filter screen.DMEM containing 20％ fetal bovine serum was added for the cultivation and passaged of the cells.The cells were purified by exchanging medium and partial enzymatic digestion.The morphology and growth status were monitored under the phase contrast microscope,and α-smooth muscle actin (α-SMA),platelet-derived growth factor receptor-β (PDGFR-β),yon Willebrand factor (vWF),glial fibrillary acidic protein (GFAP) antibodies were used for the identification of RMPs.Results RMPs migrated out of fragments after 24-48 hours of plating.On day 7,RMPs appeared in primary cultures as loose colonies.The cells reached confluence to about 80％-90％ on day 14-16.The subcultures grew faster than the primary and reached confluence on day 12-14.The culture showed typical morphology of pericyte with large irregular triangular cell body and multiple long processes,and they could be repeatedly passaged 9 times without obvious loss of characteristic phenotype.Fluorescence assay exhibited that 96％ of the cells showed positive immunofluorescence for α-SMA and PDGFR-β,confirming the purity of RMPs in culture.However,only a few of them were positive for GFAP and the cells response for vWF was absent.Conclusions High purity of rat RMPs can be obtained easily by our method without high cost-consuming.Hcrc wc cstablished a simple mcthod for the primary culture of rat RMPs.

Mesenchymal stem cells: environmentally responsive therapeutics for regenerative medicine

Mesenchymal stem cells (MSCs) are partially defined by their ability to differentiate into tissues including bone, cartilage and adipose in vitro, but it is their trophic, paracrine and immunomodulatory functions that may have the greatest therapeutic impact in vivo. Unlike pharmaceutical treatments that deliver a single agent at a specific dose, MSCs are site regulated and secrete bioactive factors and signals at variable concentrations in response to local microenvironmental cues. Significant progress has been made in understanding the biochemical and metabolic mechanisms and feedback associated with MSC response. The anti-inflammatory and immunomodulatory capacity of MSC may be paramount in the restoration of localized or systemic conditions for normal healing and tissue regeneration. Allogeneic MSC treatments, categorized as a drug by regulatory agencies, have been widely pursued, but new studies demonstrate the efficacy of autologous MSC therapies, even for individuals affected by a disease state. Safety and regulatory concerns surrounding allogeneic cell preparations make autologous and minimally manipulated cell therapies an attractive option for many regenerative, anti-inflammatory and autoimmune applications.