| Background:Bone marrow-derived mesenchymal stem cells (MSC) are adult stem cells that reside within the bone marrow compartment. MSC possess a multi-lineage differentiation potential and have the capability to differentiate into and rebuild various tissues. MSC have become the recent focus of intense research in the treatment of ischemic disease due to their ability to repair and rebuild injured vessels. Brain microvascular endothelial cells (BMEC) are the major cellular component of the blood brain barrier (BBB), which separates the brain microenvironment from circulating blood. BMEC are characterized by their ability to form tight junctions, low number of pinocytotic vesicles, and the presence of specialized transport systems that are responsible for the maintenance of the ionic and metabolic homeostasis of the brain parenchyma. In ischemic cerebrovascular disease, the injury of BMEC induces the opening of the BBB, which may lead to a brain edema and nerve cell damage, and the recovery and neogenesis of ischemic penumbral microvasculature is a key point in the retrieval of injured nerve cells. Currently, there is interest in the use of stem cells to repair injured nerve tissue as therapy for ischemic cerebrovascular disease. However, experimental data on the effects of stem cells on injured brain microvasculature and the BBB are limited. We incubated BMEC under hypoxic conditions in order to model the pathophysiological conditions of the brain microvasculature and the BBB following ischemic cerebrovascular disease and to observe the effects of BMEC on the differentiation of co-cultured MSC. In addition, we investigated the paracrine actions of MSC on BMEC, including their proliferation, migration and monolayer permeability. Our data may provide basic experimental evidence for the application of MSC in ischemic cerebrovascular disease.Objectives: 1. To isolate, culture and identify rat BMEC and human MSC. To establish indirect and direct co-cultures model of BMEC and MSC. To explore the actions of BMEC to indirectly and directly co-cultured MSC under hypoxic conditions.2. To measure the content of vascular endothelial growth factor (VEGF) and matrix metalloproteinases-9 (MMP-9) in BMEC and MSC conditioned media. To study the paracrine actions of MSC to proliferation, migration and the permeability of BMEC under hypoxic conditions.Methods:1. To isolate, culture and identify rat BMEC and human MSC: We used twice enzymatic digestion(0.1% type II collagenase and collagenase/dispase) and density gradient centrifugation to isolate purified brain microvascular fragments, which were then plated on collagen type IV and fibronectin coated plastic culture flasks and were cultured in DMEM containing 20% FCS, basic fibroblast growth factor (bFGF, 10 ng/ml), heparin (100μg/ml). BMEC were identified by immunocytochemistry of vWF. We used density gradient centrifugation to isolate MSC, which were identified by flow cytometry for CD29, CD34, CD44, CD105 and Flk-1. To establish indirect and direct co-cultures model: to induce hypoxia, cultured cells were placed in a modular incubator chamber flushed with a gas mixture of 2%O2–5%CO2–93%N2. Indirect co-cultures were established using Millicell Culture Plate Inserts. BMEC and MSC were suspended in DMEM-10 and planted in the upper and lower chambers, respectively, of 24-well plates (5×103 cells/well). A control experiment was performed to examine the role of VEGF in isolation, 10μg/ml anti-VEGF polyclonal antibody was added to both the upper and lower chambers. Direct co-cultures were established by seeding two different cell types together at ratios of 5000:5000 cells/ml onto tissue culture flasks suspended in DMEM-10. Indirectly and directly co-cultures were incubated under normal or hypoxic conditions for 5 days, and MSC differentiation was analyzed using flow cytometry (quantitation detection of Flk-1) and fluorescence immunocytochemistry (qualitation detection of Flk-1 and vWF).2.①To measure the content of VEGF and MMP-9 in BMEC and MSC conditioned media: BMEC and MSC conditioned media under normal and hypoxic conditions (termed BMECCM N and MSCCM N, BMECCM H and MSCCM H , respectively) were collected, the contents of VEGF and MMP-9 in the conditioned media were confirmed using ELISA assays.②To study the paracrine actions of MSC to proliferation and migration of BMEC under hypoxic conditions: BMEC were cultured in different conditioned media (DMEM-10, MSCCM N, MSCCM H, boiled MSCCM H, MSCCM H added anti-VEGF antibody, MSCCM H added MMP-9 inhibitor I). The effects of different conditioned media on BMEC proliferation and migration were assayed using Cell Counting Kit-8 and Transwell culture system under hypoxic conditions, respectively.③To study the paracrine actions of MSC to the permeability of BMEC: the effects of different conditioned media on the permeability of the BMEC monolayer were assayed using the transendothelial electrical resistance(TEER).Results:1. MSC typically expressed the antigens CD105, CD29, and CD44, but were negative for Flk-1 and CD34; BMEC typically expressed the endothelial characteristics of vWF. Both the indirectly and directly co-cultured cells grew well under normoxic or hypoxic conditions.The freshly isolated MSC did not express Flk-1 or vWF. After normoxic indirect co-culture with BMEC for 5 days, MSC were still negative for Flk-1; while indirect co-culture under hypoxic conditions induced approximately 7.58±0.58% (n=6, P<0.05) of MSC to express Flk-1, fluorescence immunocytochemistry also confirmed that a few cells expressed Flk-1, but vWF staining was still negative. In the direct co-culture group, after 5 days of direct co-culturing in normoxic and hypoxic conditions, respectively, MSC expressing Flk-1 accounted for 13.76±1.67% (n=6, P<0.001) and 23.64±2.50% (n=6, P<0.001) of the total co-cultured cell population, which was a significant difference (n=6, P<0.001). Fluorescence immunocytochemistry also confirmed that some cells had begun to express Flk-1, though none of the Flk-1 positive cells coexpressed vWF in the normoxic direct co-cultures. Importantly, some Flk-1 positive cells began to coexpress vWF simultaneously in the hypoxic direct co-cultures.2.①To measure the content of VEGF and MMP-9 in BMEC and MSC conditioned media: compared with normoxia, hypoxia induced an increase in VEGF secretion in both the BMEC and MSC conditioned media. The amount of VEGF in the MSCCM H was much higher than the BMECCM H. We did not detect MMP-9 in the BMECCM N. The contents of MMP-9 in the MSCCM H were much higher than the BMECCM H and the MSCCM N.②To study the paracrine actions of MSC to proliferation and migration of BMEC under hypoxic conditions: compared to DMEM-10, MSCCM H significantly enhanced BMEC proliferation. In addition, the effect of MSCCM H was much stronger than MSCCM N (optical density (OD) values: 0.947±0.103 versus 0.532±0.028, P<0.001, n=6). The proliferative effect of MSCCM H was completely abolished by boiling. As expected, the effect of MSCCM H was inhibited by the addition of a VEGF-blocking antibody (OD values: 0.947±0.103 versus 0.419±0.034, P<0.001, n=6), while results indicated that the MMP-9 inhibitor did not significantly attenuate proliferation (OD values: 0.947±0.103 versus 0.902±0.065, P=0.963, n=6). MSCCM H induced a significant increase in BMEC migration compared to DMEM-10 and MSCCM N (238±27 versus 154±24, P<0.01, n=6). As with BMEC proliferation, boiling eliminated the chemoattractant properties of MSCCM H, and the VEGF-blocking antibody partly attenuated migration (238±27 versus 150±20, P<0.001, n=6). However, the MMP-9 inhibitor significantly inhibited migration (238±27 versus 106±18, P<0.001, n=6).③To study the paracrine actions of MSC to the permeability of BMEC: normoxic controls maintained in a standard incubator environment showed no significant change in the TEER over the 24-hour period. While the TEER of hypoxic controls decreased significantly from 6 h to 18 h, with the greatest decrease detected at 18 h (77.2±1.8% of original). Under hypoxic conditions, MSCCM H caused a sharp drop within the first 2 h, with the greatest decrease detected at 2 h (50.5±2.6% of original). Thereafter, resistances began to recover until 24 h, but remained at a low level. The anti-VEGF antibody and MMP-9 inhibitor partially inhibited the decrease, with the greatest decrease detected at 2 h (60.3±3.6% of original) and 3 h (76.0±2.4% of original) separately.Conclusions:1. We have isolated, cultured and identified rat BMEC and human MSC successfully, and have established indirect and direct co-cultures model successfully. BMEC cannot induce MSC to differentiate to endothelial cells through paracrine under normoxic conditions, BMEC can induce MSC to begin to differentiate to endothelial cells through direct cell contact. Hypoxia is important for MSC to differentiate into endothelial cells. Directly co-cultured under hypoxic conditions, BMEC can induce more MSC to differentiate to endothelial cells, and more thoroughly.2.①The amount of VEGF and MMP-9 in the MSC conditioned media was much higher than the BMEC conditioned media. Compared with normoxia, hypoxia induced an increase in VEGF secretion in both the BMEC and MSC conditioned media.②MSC can enhance the proliferation and migration of BMEC significantly through paracrine, MMP-9 paracrined by MSC is important for migration of BMEC, and VEGF is momentous for both proliferation and migration of BMEC.③MSC can increase the permeability of the BMEC monolayer strikingly through paracrine, it is important to investigate MSC secreting high levels of MMP-9 and VEGF, which induce an increase in the permeability of the BMEC monolayer.
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