Objective: To investigate whether tanshinone ⅡA can protect the apoptosis of mice cochlear pericytes induced by high glucose and its specific protective mechanism, so as to provide experimental evidence for the prevention and treatment of diabetic hearing loss. Methods: C57BL/6J male mice were used to prepare type 2 diabetes model, which were divided into normal (NG) group, diabetic (DM) group, diabetic+tanshinone ⅡA (HG+tanshinone ⅡA) group and tanshinone ⅡA group. Each group had 10 animals. Primary cochlear pericytes were divided into NG group, HG group (high glucose 35 mmol/L), HG+tanshinone ⅡA (1, 3, 5 μmol/L) group, HG+Tanshinone ⅡA+LY294002 (PI3K/AKT pathway inhibitor) group, LY294002 group, tanshinone ⅡA group and DMSO group. Auditory brainstem response (ABR) was used to measure hearing threshold. Evans blue was used to detect the permeability of blood labyrinth barrier in each group. TBA methods were used to detect oxidative stress levels in various organs of mice. Morphological changes of stria vascularis were observed by hematoxylin-eosin staining (HE). Evans blue was used to detect the vascular labyrinth barrier permeability in cochlea. The expression of apoptosis protein in stria vascularis pericytes was observed by immunofluorescence. Pericytes apoptosis rate was observed by flow cytometry. DCFH-DA was combined with flow cytometry to detect intracellular ROS content, and Western blot was used to detect the expression of apoptotic proteins (Cleaved-caspase3, Bax), anti-apoptotic proteins (BCL-2) and pathway proteins (PI3K, p-PI3K, AKT, p-AKT). SPSS software was used for statistical analysis. Independent sample t test was performed, and P<0.05 was considered statistically significant. Results: Animal experiments: Tanshinone ⅡA decreased the hearing threshold of DM group [(35.0±3.5) dB SPL vs. (55.3±8.1) dB SPL] (t=4.899, P<0.01), decreased the oxidative stress level in cochlea (t=4.384, P<0.05), improved the structure disorder, atrophy of cochlea vascular lines, vacuole increased phenomenon. Tanshinone ⅡA alleviated the increased permeability of the blood labyrinth barrier [Evans blue leakage (6.84±0.27) AU vs. (8.59±0.85) AU] in the cochlea of DM mice (t=2.770, P<0.05), reversed the apoptotic protein: Caspase3 (t=4.956, P<0.01) and Bax (t=4.388, P<0.05) in cochlear vascularis. Cell experiments: Tanshinone ⅡA decreased intracellular ROS content in a concentration-dependent way (t=3.569, P<0.05; t=4.772, P<0.01; t=7.494, P<0.01); Tanshinone ⅡA decreased apoptosis rate and apoptotic protein, and increased the expression of anti-apoptotic protein, p-PI3K/PI3K and p-AKT/AKT in concentration-dependent manner (all P values<0.05); LY294002 reversed the protective effect of tanshinone ⅡA on pericytes apoptosis (all P values<0.05). Conclusion: Tanshinone ⅡA can inhibit the apoptosis of cochlear pericytes induced by high glucose by reducing oxidative stress level and activating PI3K/AKT signaling pathway under high glucose environment, thus playing a protective role in diabetic hearing loss.
Animals ; Male ; Mice ; Apoptosis ; bcl-2-Associated X Protein ; Diabetes Mellitus, Type 2 ; Evans Blue ; Glucose ; Hearing Loss ; Mice, Inbred C57BL ; Pericytes/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Reactive Oxygen Species/metabolism* ; Signal Transduction

The brain pericyte is a unique and indispensable part of the blood-brain barrier (BBB), and contributes to several pathological processes in traumatic brain injury (TBI). However, the cellular and molecular mechanisms by which pericytes are regulated in the damaged brain are largely unknown. Here, we show that the formation of neutrophil extracellular traps (NETs) induces the appearance of CD11b⁺ pericytes after TBI. These CD11b⁺ pericyte subsets are characterized by increased permeability and pro-inflammatory profiles compared to CD11b^- pericytes. Moreover, histones from NETs by Dectin-1 facilitate CD11b induction in brain pericytes in PKC-c-Jun dependent manner, resulting in neuroinflammation and BBB dysfunction after TBI. These data indicate that neutrophil-NET-pericyte and histone-Dectin-1-CD11b are possible mechanisms for the activation and dysfunction of pericytes. Targeting NETs formation and Dectin-1 are promising means of treating TBI.
Blood-Brain Barrier/metabolism* ; Brain/pathology* ; Brain Injuries, Traumatic/metabolism* ; Extracellular Traps/metabolism* ; Histones ; Humans ; Lectins, C-Type ; Pericytes/pathology*

Objective: To study the changes in the permeability of the blood labyrinth barrier of the aging cochlea in mice, and to establish a non-contact co-culture model of endothelial cells (EC) and pericytes (PC) to furtherly investigate the cochlear stria vascularis microvascular pericytes impact on the permeability of endothelial cells. Methods: C57BL/6J mice were divided into two groups, three months old as young group, 12 months old as senile group. Cell experiment was divided into four groups, EC group, EC+PC co-culture group, D-gal+EC group and D-gal+EC+PC co-culture group. Auditory brainstem response (auditory brain response, ABR) was used to detect the auditory function of the two groups of mice. Evans blue staining was applied to detect the permeability of the cochlear blood labyrinth barrier of the two groups of mice. Transmission electron microscopy was used to observe the ultrastructure of blood labyrinth barrier endothelial cells, pericytes and tight junctions in the two groups of mice. Immunohistochemistry was used to detect the expression levels of tight junction proteins in the stria vascularis of the cochlea of the two groups of mice. Transwell chamber was used to detect the permeability of endothelial cells. Western blot and immunofluorescence technology were used to detect the expression level of tight junction protein on endothelial cells. SPSS 20.0 software was used to analyze the data. Results: Compared with the young group, the ABR threshold of the aging group was significantly increased, the latency of wave I was prolonged (t=10.25, P<0.01;t=5.61, P<0.05), the permeability of the cochlear blood labyrinth barrier was increased and the expression of tight junction protein on the vascular stria was decreased (P<0.05). The cochlear ultrastructure showed that the cochlear vascular stria microvascular lumen was deformed, the basement membrane thickened and the tight junction gap between endothelium enlarged. The positive rate of ECs and PCs in primary culture was more than 95%. The cells induced by 15 g/L D-gal were determined to be senescent cells. Compared with EC group, the expression of tight junction protein in endothelial cells of D-gal+EC group decreased（t=7.42，P<0.01；t=13.19，P<0.05）and the permeability increased (t=11.17, P<0.01). In the co-culture group, the expression of tight junction protein between endothelial cells in EC+PC co-culture group and D-gal+EC+PC co-culture group increased and the permeability decreased. Conclusions: In aging mice, the permeability of cochlear blood labyrinth barrier will increase and the level of tight junction protein will decrease; in aging state, cochlear vascular stria microvascular pericytes may affect endothelial cell permeability by regulating the expression of tight junction protein.
Animals ; Cochlea ; Endothelial Cells ; Mice ; Mice, Inbred C57BL ; Pericytes ; Permeability ; Stria Vascularis ; Tight Junctions

Objective: To investigate whether large conductance calcium-activated potassium channel (BK(Ca)) was involved in the migration of pericytes (PC) in the mice of senile cochlear stria vascularis capillaries PC. Methods: C57BL/6J mice were divided into 3-month (n=10) and 12-month groups (n=10). Auditory brainstem response (ABR) was used to test the hearing threshold of each group. The immunofluorescence was used to detect the expression changes of osteopontin (OPN) and β-BK(Ca) channels on cochlear stria vascularis PC. The morphological changes of perivascular cells in cochlea were observed by transmission electron microscope (TEM). Cell experiment: The PC, which were in the stria vascularis of the cochlea were primary cultured and identified. A cell senile model was made with D-gal. The appropriate intervention concentration of low galactose (D-gal) was determined by CCK8. β-galactosidase (SA-β-gal) staining was used to evaluate the cell decrept level. The change of BK(Ca) channels current on PC were recorded by whole cell patch clamp technique. The expression of BK(Ca) channels on PC was detected by immunofluorescence. The migration and invasion ability of two groups were detected by using Scratch test and Transwell. The levels of OPN and β-BK(Ca) channels were detected by Western blot. SPSS 22.0 software was used to analyze the data. Results: The ABR threshold in the 12-month group was higher than 3-month group (t=12.66, P<0.01). In the 12-month group, the expression of β-BK(Ca) channel was lower and the expression of OPN was increased (t=14.64, P<0.01; t=20.73, P<0.01). In TEM, cochlear stria vascularis PC were tightly connected to endothelial cells in 3-month group, while PC were loosely connected to endothelial cells or PC soma were separated from the capillary in 12-month group. Cell experiment: The positive rate of PC in the primary cultured cochlear stria vascularis is above 95%. Compared with the SA-β-gal stained cells in the control group, the positive rate of 15 mg/ml D-gal intervention PC was 85% (t=36.90, P<0.01). Whole cell patch clamp BK(Ca) channels current decreased in the D-gal group compared with the young group PC (t=12.18, P<0.05). The OPN expression in the senile group was higher than control group (t=16.30, P<0.01), while the β-BK(Ca) channels expression was decreased (t=11.98, P<0.01; t=15.72, P<0.05), and migration ability raised (t=7.91, P<0.01;t=7.59, P<0.01). After intervened of BK(Ca) channels specific blocker IBTX in the D-gal group, the expression of OPN and migration were increased (t=4.26, P<0.05; t=5.88, P<0.01; t=21.97, P<0.01). Conclusion: PC migration capacity were increased during the senile period, and the expression of β-BK(Ca) channel was decreased. The administration of IBTX, a specific blocker of BK(Ca) channel, at the cell level could increase the migration capacity, suggesting that BK(Ca) might be involved in the migration of PC in the stria vascularis of the aging cochlea.
Aging ; Animals ; Cochlea ; Endothelial Cells ; Large-Conductance Calcium-Activated Potassium Channels ; Mice ; Mice, Inbred C57BL ; Pericytes ; Stria Vascularis

pericytes from human corpus cavernosum tissue and to evaluate the angiogenic ability of the human cavernous EC or pericytes for the study of high glucose-induced angiopathy.MATERIALS AND METHODS: For primary human cavernous EC culture, cavernous tissues were implanted into Matrigel in dishes. For primary human cavernous pericyte culture, cavernous tissues were settled by gravity into dishes. We performed immunocytochemistry and Western blot to determine phenotype and morphologic changes from passage 1 to 5. The primary cultured cells were exposed to a normal-glucose (5 mmol/L) or a high-glucose (30 mmol/L) condition, and then tube formation assay was done.RESULTS: We successfully isolated high-purity EC and pericytes from human corpus cavernosum tissue. Primary cultured EC showed highly positive staining for von Willebrand factor, and pericyte revealed positive staining for NG2 and platelet-derived growth factor receptor-β. Primary cultured EC and pericytes maintained their cellular characteristics up to passage 2 or 3. However, we observed significant changes in their typical phenotype from the passage 4 and morphological characteristics from the passage 3. Human cavernous EC or pericytes formed well-organized capillary-like structures in normal-glucose condition, whereas severely impaired tube formation was detected in high-glucose condition.CONCLUSIONS: This study provides a simple and nonenzymatic method for primary culture of human cavernous EC and pericytes. Our study will aid us to understand the pathophysiology of diabetic erectile dysfunction, and also be a valuable tool for determining the efficacy of candidate therapeutic targets.]]>
Blotting, Western ; Cells, Cultured ; Diabetes Mellitus ; Endothelial Cells ; Erectile Dysfunction ; Gravitation ; Humans ; Immunohistochemistry ; Male ; Methods ; Pericytes ; Phenotype ; Platelet-Derived Growth Factor ; von Willebrand Factor

Cerebral pericytes are perivascular cells that stabilize blood vessels. Little is known about the plasticity of pericytes in the adult brain in vivo. Recently, using state-of-the-art technologies, including two-photon microscopy in combination with sophisticated Cre/loxP in vivo tracing techniques, a novel role of pericytes was revealed in vascular remodeling in the adult brain. Strikingly, after pericyte ablation, neighboring pericytes expand their processes and prevent vascular dilatation. This new knowledge provides insights into pericyte plasticity in the adult brain.
Animals ; Brain ; blood supply ; physiology ; physiopathology ; Brain Diseases ; physiopathology ; Capillaries ; physiology ; Cellular Microenvironment ; Diabetic Retinopathy ; physiopathology ; Endothelial Cells ; physiology ; Humans ; Pericytes ; physiology ; Vascular Remodeling

Objective To observe the change pattern of pericyte number at different time periods after mice skeletal muscle contusion and discuss its role in wound age estimation. Methods A mice gastrocnemius muscle contusion model was established. The form and number changes of pericytes at 1, 3, 5, 7, 9, 14, and 28 d post-injury were detected by multiple immunofluorescence staining. Results Compared with the slender shape of pericytes in normal skeletal muscles, pericytes in the contusion area had increased volume, rounder form and a round nuclei. Part of pericytes were found to express satellite cell markers paired-box transcription factor （Pax7） or myoblast determination 1 （MyoD1）. The changes of pericyte number in skeletal muscles after contusion were time-dependant, and showed unimodal distribution with the extension of wound age. In the central contusion area, the number of pericytes peaked at 5 d post-injury while in the peripheral contusion area, the number of pericytes peaked at 5 d and 7 d post-injury. Conclusion The number of pericytes in contusion area varies time-dependently after skeletal muscle contusion in mice and might be a reference index for muscle wound age estimation, and is involved in the repair and regeneration of skeletal muscle injury.
Animals ; Contusions ; Disease Models, Animal ; Mice ; Muscle, Skeletal ; Pericytes ; Rats, Sprague-Dawley

PURPOSE: To evaluate the relationship between pericytes and endothelial cells in retinal neovascularization through histological and immunofluorescent studies. METHODS: C57BL/6J mice were exposed to hyperoxia from postnatal day (P) 7 to P12 and were returned to room air at P12 to induce a model of oxygen-induced retinopathy (OIR). The cross sections of enucleated eyes were processed with hematoxylin and eosin. Immunofluorescent staining of pericytes, endothelial cells, and N-cadherin was performed. Microfluidic devices were fabricated out of polydimethylsiloxane using soft lithography and replica molding. Human retinal microvascular endothelial cells, human brain microvascular endothelial cells, human umbilical vein endothelial cells and human placenta pericyte were mixed and co-cultured. RESULTS: Unlike the three-layered vascular plexus found in retinal angiogenesis of a normal mouse, angiogenesis in the OIR model is identified by the neovascular tuft extending into the vitreous. Neovascular tufts and the three-layered vascular plexus were both covered with pericytes in the OIR model. In this pathologic vascularization, N-cadherin, known to be crucial intercellular adhesion molecule, was also present. Further evaluation using the microfluidic in vitro model, successfully developed a microvascular network of endothelial cells covered with pericytes, mimicking normal retinal angiogenesis within 6 days. CONCLUSIONS: Pericytes covering endothelial cells were observed not only in vasculature of normal retina but also pathologic neovascularization of OIR mouse at P17. Factors involved in the endothelial cell-pericyte interaction can be evaluated as an attractive novel treatment target. These future studies can be performed using microfluidic systems, which can shorten the study time and provide three-dimensional structural evaluation.
Animals ; Brain ; Cadherins ; Endothelial Cells ; Eosine Yellowish-(YS) ; Fungi ; Hematoxylin ; Human Umbilical Vein Endothelial Cells ; Humans ; Hyperoxia ; In Vitro Techniques ; Lab-On-A-Chip Devices ; Mice ; Microfluidics ; Microvessels ; Neovascularization, Pathologic ; Pericytes ; Placenta ; Retina ; Retinal Neovascularization ; Retinaldehyde

CD146 Antigen ; metabolism ; Humans ; Pericytes ; cytology ; metabolism ; Receptor, Platelet-Derived Growth Factor beta ; metabolism

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.
Drugs, Chinese Herbal ; pharmacology ; Fibrosis ; Humans ; Kidney ; cytology ; drug effects ; pathology ; Myofibroblasts ; cytology ; Pericytes ; cytology ; Receptors, Platelet-Derived Growth Factor ; metabolism ; Signal Transduction ; Vascular Endothelial Growth Factor A ; metabolism