Supportive angiogenic and osteogenic differentiation of mesenchymal stromal cells and endothelial cells in monolayer and co-cultures

Sites of implantation with compromised biology may be unable to achieve the required level of angiogenic and osteogenic regeneration. The specific function and contribution of different cell types to the formation of prevascularized, osteogenic networks in co-culture remains unclear. To determine how bone marrow-derived mesenchymal stromal cells （BMSCs） and endothelial cells （ECs） contribute to cellular proangiogenic differentiation, we analysed the differentiation of BMSCs and ECs in standardized monolayer, Transwell and co-cultures. BMSCs were derived from the iliac bone marrow of five patients, characterized and differentiated in standardized monolayers, permeable Transwells and co-cultures with human umbilical vein ECs （HUVECs）. The expression levels of CD31, von Willebrand factor, osteonectin （ON） and Runx2 were assessed by quantitative reverse transcriptase polymerase chain reaction. The protein expression of alkaline phosphatase, ON and CD31 was demonstrated via histochemical and immunofluorescence analysis. The results showed that BMSCs and HUVECs were able to retain their lineage-specific osteogenic and angiogenic differentiation in direct and indirect co-cultures. In addition, BMSCs demonstrated a supportive expression of angiogenic function in co-culture, while HUVEC was able to improve the expression of osteogenic marker molecules in BMSCs.

Co-culture of Mesenchymal Stem Cells with Umbilical Vein Endothelial Cells under Hypoxic Condition

By co-culturing humm mesenchymal stem cells (hMSCs) and human umbilical rein endothelial cells (HUVECs) under hypoxia and creating a microenvironment similar to that of transplanted hMSCs for the treatment of avascular ni ANFH, the effect of hMSCs on survival, apoptosis, migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) under the hypoxic condition were investigated in vitro. hMSCs and HUVECs were cultured and identified in vitro. Three kinds of conditioned media, CdM-CdMNOR, CdM-CdMHYP and HUVEC-CdMHYP were prepared. HUVECs were cultured with these conditioned media under hypoxia. The survival rate, apoptosis rate, migration and angiogenesis of HUVECs were respectively detected by CCK-8, flow cytometry, Transwell and tube formation assay. The content of SDF-1α, VEGF and IL-6 in CdM was determined by ELISA. Our results showed that hMSCs and HUVECs were cultured and identified successfully. Compared with MSC-CdMNOR and HUVEC-CdMHYP groups, the survival rate, migra-tion and angiogenesis of HUVECs in MSC-CdMHYP group were significantly increased while the apoptosis rate was declined (P<0.05). Moreover, the expression of SDF-1α, VEGF and IL-6 in MSC-CdMHYP group was up-regulated. Under hypoxia, the apoptosis of HUVECs was inhibited while survival, migration and angiogenesis were improved by co-culture of hMSCs and HUVECs. The underlying mechanism may be that hMSCs could secrete a number of cytokines and improve niche, which might be helpful in the treatment of femoral head necrosis.

Differences in the primary culture, purification and biological characteristics between endothelial cells and smooth muscle cells from rat aorta

Objective： To investigate the differences of primary culture, purification and biological characteristics between endothelial cells and smooth muscle cells from rat aorta. Methods： Endothelial cells were obtained using the vascular ring adherence, collagenase digestion method and an improved vascular ring adherence method, while smooth muscle cells were separated from tissue sections of rat aorta. Clones of endothelial cells were selected by limiting dilution assay. Both cell types were identified using specific cell immunofluorescent markers, and phase contrast microscopy was used to observe the morphological disparity between endothelial cells and smooth muscle cells at the single cell and colony level. Cell proliferation was determined by the cell counting kit-8. Differences between endothelial cells and smooth muscle cells were evaluated in trypsin digestion time, attachment time and recovery after cryopreservation. Results： Endothelial cells were obtained by all three methods. The improved vascular ring method provided the most reproducible results. Cells were in good condition, and of high purity. Smooth muscle cells were cultured successfully by the tissue fragment culture method. Clonal expansion of single endothelial cells was attained. The two cell types expressed their respective specific markers, and the rate of proliferation of smooth muscle cells exceeded that of endothelial cells. Endothelial cells were more sensitive to trypsin digestion than smooth muscle cells. In addition, they had a shorter adherence time and better recovery following cryopreservation than smooth muscle cells. Conclusion： The improved vascular ring method was optimal for yielding endothelial cells. Limiting dilution is a novel and valid method for purifying primary endothelial cells from rat aorta. Primary rat endothelial cell and vascular smooth muscle cell cultures exhibited different morphological characteristics, proliferation rate, adherence time, susceptibility to trypsin digestion and recovery after cryopreservation. Our research can be a good foundation for further application in the regeneration of blood vessel.

Determinants of PHGPx Expression in a Cultured Endothelial Cell Line

Selenoprotein biosynthesis may not only be affected by the availability of selenium and the transcription rate of pertinent genes but also by the activity of components of the selenocysteine incorporation complex, SelA, B, C, or D. Incorporation of selenocysteine into selenoproteins requires a complex co-translational mechanism guaranteeing the correct recoding of the termination codon TGA as selenocysteine codon. A particular tRNASer(Sec) is enzyrnatically transformed by selenophosphate into tRNAsec which recognizes the UGA codon by means of a specific elongation factor (SelB) and a peculiar mRNA secondary structure. Selenophosphate is formed from selenide and ATP by the SelD gene product, selenophosphate synthase (SelD). To further elucidate the biological role of phospholipid hydroperoxide GPx (PHGPx), we transformed cells with a heterologous (pig) PHGPx gene and/or an additional (human) SelD gene and studied the behaviour of these cells under selenium depletion and repletion. Transfection of the endothelial cell line ECV 304 with either PHGPx cDNA or SelD cDNA did not result in a substantial increase of PHGPx activities, independent of selenium supply. However, cells co-trans fected with both, PHGPx and SelD cDNA, expressed significantly higher PHGPx activlty. This effect was much more pronounced under selenium limiting conditions. The enhanced PHGPx activity correlated with two functional pararneters, increased capability to reduce hydroperoxides and less sensitivity against H2O2-induced cytotoxicity. Thus, the ECV cells, stably transfected with PHGPx and SelD cDNA, provide a model to specifically investigate the role of PHGPx in endothelial cell function

Effects of rotational culture on morphology,nitric oxide production and cell cycle of endothelial cells

Devices for the rotational culture of cells and the study of biological reactions have been widely applied in tissue engineering.However,there are few reports exploring the effects of rotational culture on cell morphology,nitric oxide(NO)production,and cell cycle of the endothelial cells from human umbili-cal vein on the stent surface.This study focuses on these parameters after the cells are seeded on the stents.Results showed that covering of stents by endothelial cells was improved by rotational culture.NO produc-tion decreased within 24 h in both rotational and static culture groups.In addition,rotational culture signifi-cantly increased NO production by 37.9%at 36 h and 28.9%at 48 h compared with static culture.Flow cytometry showed that the cell cycle was not obviously influenced by rotational culture.Results indicate that rotational culture may be helpful for preparation of cell-seeded vascular grafts and intravascular stents,which are expected to be the most frequently implanted materials in the future.

Primary culture and identification about brain microvascular endothelial cells of rabbits

Objective:To establish a simple and efficient culture method of primary rabbit brain microvascular endothelial cells,provide important carriers and tool cells for the research of related cerebrovascular diseases.Methods:The cerebral cortexes of rabbits were collected aseptic and inoculated after cutting,passing through cell sieve,bovine serum albumin density gradient centrifugation,typeⅡcollagenase digestion,finally inoculated and cultured.The cultured cells were identified by cell morphological observation and angiogenesis experiment.Results:Under the inverted microscope,the cells were short fusiform or polygonal,and grew in clusters and adhere to the wall.After the cells were densely fused,they would be in a typical monolayer flat,“pebbled"mosaic arrangement.Tube formation test had the ability to form tubes structure.Conclusion:This method can successfully separate and cultivate primary rabbit brain microvascular endothelial cells.

Discussion on Serum Technique and Cryopreservation Technique in the Culture of Liver Sinusoidal Endothelial Cells

Hepatic sinusoidal endothelial cells are a kind of highly differentiated cells in hepatic sinusoids, which play an important role in the occurrence and development of liver fibrosis. Hepatic sinusoidal endothelial cells are often used in the study of liver fibrosis. In the process of culturing liver sinusoidal endothelial cells, the treatment of serum for culture and cryopreservation of cells are relatively complicated and error-prone. If the technical details of serum treatment and cell cryopreservation are not handled properly, it will have a more obvious effect on the effect of hepatic sinusoidal endothelial cell culture. We have gained experience in the theoretical study of liver fibrosis and the operation of cell culture experiments, and this paper summarized the details of liver sinusoidal endothelial cell culture such as the problems of serum preservation, thawing, precipitates and heat inactivation, as well as methods and precautions for cell cryopreservation.

Nerve Growth Factor Modulate Proliferation of Cultured Rabbit Corneal Endothelial Cells and Epithelial Cells

Summary： In order to investigate the effect of nerve growth factor （NGF） on the proliferation of rabbit corneal endothelial cells and epithelial cells, the in vitro cultured rabbit corneal endothelial cells and epithelial cells were treated with different concentrations of NGF.MTT assay was used to examine the clonal growth and proliferation of the cells by determining the absorbency values at 570 nm. The results showed that NGF with three concentrations ranging from 5 U/mL to 500 U/mL enhanced the proliferation of rabbit corneal endothelial cells in a concentration-dependent manner. 50 U/mI. and 500 U/mI. NGF got more increase of proliferation than that of 5 U/mL NGF did. Meanwhile, 50 U/mL and 500 U/mL NGF could promote the proliferation of the rabbit corneal epithelial cells significantly in a concentration-dependent manner. However, 5 U/mL NGF did not enhance the proliferation of epithelial cells. It was suggested that exogenous NGF can stimulate the proliferation of both rabbit corneal endothelial and epithelial cells, but the extent of modulation is different.

Proliferation and Differentiation of Neural Stem Cells Co-Cultured with Cerebral Microvascular Endothelial Cells after Oxygen-glucose Deprivation

Various stem cells, including neural stem cells （NSCs）, have been extensively studied in stroke models, but how to increase neuronal differentiation rate of NSCs remains unresolved, particu- larly in a damaged environment. The purpose of this study was to investigate the effects of cerebral mi- crovascular endothelial cells （CMECs） on the neurogenesis of NSCs with or without oxygen-glucose deprivation （OGD）. The NSCs acquired from primary culture were immunostained to prove cell purity. Survival and proliferation of NSCs were determined after the co-culture with CMECs for 7 days. After removing the CMECs, NSCs were randomly divided into two groups as follows： OGD and non-OGD groups. Both groups were maintained in differentiation culture for 4 days to evaluate the differentiation rate. Mouse embryo fibroblast （MEF） cells co-cultured with NSCs served as control group. NSCs co-cultured with CMECs had an increase in size （on the 7th day： 89.80±26.12 μm vs. 73.08±15.01μm, P〈0.001） （n=12） and number [on the 7th day： 6.33±5.61/high power objective （HP） vs. 2.23±1.61/HP, P〈0.001] （n=12） as compared with those co-cultured with MEF cells. After further differentiation cul- ture for 4 days, NSCs co-cultured with CMECs had an increase in neuronal differentiation rate in OGD and non-OGD groups, but not in the control group （15.16% and 16.07% vs. 8.81%; both P〈0.001） （n=6） This study provided evidence that OGD could not alter the effects of CMECs in promoting the neuronal differentiation potential of NSCs. These findings may have important implications for the development of new cell therapies for cerebral vascular diseases.

Baicalin Induces IFN-α/β and IFN-γ Expressions in Cultured Mouse Pulmonary Microvascular Endothelial Cells

We studied the effect of baicalin,an extract from Radix Scutellariae（a traditional Chinese medicine） in inducing mouse pulmonary microvascular endothelial cells（MPMVECs） to produce interferons（IFNs）.MPMVECs were cultured in vitro in the presence of different concentrations of baicalin（10,20,and 30 μg mL-1）,and the cells and the culture media were harvested at various time intervals.The proteins and mRNA levels（relative to β-actin） of IFN-α/β and IFN-γ were analyzed by RT-PCR and enzyme-linked immunosorbent assay（ELISA）.It was observed that baicalin substantially up-regulated the expression of IFN-α/β and IFN-γ.In all baicalin-treated groups,the relative levels of IFN-α/β and IFN-γ mRNAs peaked after 12 h of culturing,and IFN-α/β and IFN-γ proteins peaked after 24 h of culturing.These results suggest that baicalin can effectively induce the expression of IFNs in pulmonary microvascular endothelial cells,and thus potentially act as an antiviral compound.This study may provide background information for developing new antiviral drugs based on baicalin.

Advances in tumor-endothelial cells co-culture and interaction on microfluidics

The metastasis in which the cancer cells degrade the extracellular matrix （ECM） and invade to the sur- rounding and far tissues of the body is the leading cause of mortality in cancer patients, With a lot of advancement in the field, yet the biological cause of metastasis are poorly understood, The microfluidic system provides advanced technology to reconstruct a variety of in vivo-like environment for studying the interactions between tumor ceils （TCs） and endothelial ceils （ECs）. This review gives a brief account of both two-dimensional models and three-dimensional microfluidic systems for the analysis of TCs-ECs co- culture as well as their applications to anti-cancer drug screening, Furthermore, the advanced methods for analyzing cell-to-cell interactions at single-cell level were also discussed,

A Study of the Subculture of Human Endothelial Cell From Umbilical Vein

Human endothelial cells derived from umbilical vein could besubcultured and survive 25 passage (double time 20～25 hours, 60～75 cumula-tive population doublings). An observation was performed with light mi-croscopy, electron microscopy and immunofluorescence microscopy using specificantiserum against factor Ⅷrelated antigen. It identified that the cultured cellswere endothelial cells. Medium RPMI-1640 supplemented with 20% humanserum, endothelial cell growth factor 200μg/ml, heparin 100μg/ml and gelatincoated flasks were very important conditions for long-term culture of humanendothelial cell.

Effects of L-Tetrahydropalmatine on NOSⅢ Gene Expression in Hypoxia and Cultured Porcine Cerebral Arterial Endothelial Cells during Reoxygenation

To investigate the expression of NOSⅢ mRNA and protein in cultured porcine cerebral arterial endothelial cells (CAEC) during hypoxia and reoxygenation and the effects of L-Tetrahydropalmatine (L-THP) on the gene expression of NOSⅢ in CAEC during hypoxia and reoxygenation. The cultured CAEC were divided into 5 groups: control, hypoxia, hypoxia+reoxygenation, hypoxia+L-THP and reoxygenation+L-THP groups. NOSⅢ mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). Immunocytochemistry was used to detect the level of NOSⅢ protein. The expression of NOSⅢ mRNA and protein were increased when CAEC were exposed to hypoxia for 1 h, and significantly decreased during reoxygenation 2, 6 and 12 h after 1-h of hypoxia. L-THP from 10 -8 mol/L to 10 -3 mol/L could inhibit the up-regulation of NOSⅢ gene expression during hypoxia and down-regulation of NOSⅢ gene expression during reoxygenation.

The role of heme-oxygenase-1 in pathogenesis of cerebral malaria in the co-culture model of human brain microvascular endothelial cell and ITG Plasmodium falciparum-infected red blood cells

Objective: To investigate the role of human host heme-oxygenase-1(HO-1) in pathogenesis of cerebral malaria in the in vitro model,Methods: The effect of human host HO-1 [human brain microvascular endothelial cell(HBMEC)] on hemoglobin degradation in the co-culture model of HBMEC and ITG Plasmodium falciparum-infected red cells(i RBC) through measurement of the enzymatic products iron and bilirubin,Results: Following exposure to the HO-1 inducer Co PPIX at all concentrations,the HBMEC cells apoptosis occurred,which could be prominently observed at 15 μM of 3 h exposure,In contrast,there was no significant change in the morphology in the non-exposed i RBC at all concentrations and exposure time,This observation was in agreement with the levels of the enzymatic degradation products iron and bilirubin,of which the highest levels(106.03 and 1 753.54% of baseline level,respectively) were observed at 15 μM vs,20 μM at 3 h vs,24 h exposure,For the effect of the HO-1 inhibitor Zn PPIX,HBMEC cell morphology was mostly unchanged,but significant inhibitory effect on cell apoptosis was seen at 10 μM for the exposure period of 3 h(37.17% of baseline level),The degree of the inhibitory effect as reflected by the level of iron produced was not clearly observed(highest effect at 10 μM and 3 h exposure),Conclusions: Results provide at least in part,insight into the contribution of HO-1 on CM pathogenesis and need to be confirmed in animal model.

EFFECTS OF MODULATED LDLs ON THE RELEASE OF ENDOTHELIN-1 AND PROSTACYCLIN BY ENDOTHELIAL CELLS IN CULTURE

Objective To study the releases of endothelin-1 and prostacyclin by endothelial cells in culture and to elucidate how these releases were influenced by smoke-treated low density lipoprotein. Methods We exposed en- dothelial cell cultures to native or oxidized low density lipoproteins,low density lipoproteins treated by dimethylsul- foxide-soluble particles from cigarette smoke or dimethylsulfoxide alones. The release of endothelin-1 was assayed by bioassay and the release of prostacyclin was assayed by radioimmunoassay. Results Low density lipoproteins treated by smoke significantly increased the release or endothelin-1 (P<0.025) and decreased the release of prostacyclin (P< 0.02) by endothelial cells in culture, contrast to native or dimethylsulfoxide-treated lipoproteins. Conclusion The main part or vasoconstrictor activity in conditioned medium from bovine aortic EC is endothelin-1.

EFFECT OF QUERCETIN ON CULTURED HUMAN VASCULAR ENDOTHELIAL CELLS

Objective To study the effects of quercetin (Que) on the release of endothelin-1 (ET-1) and prostacylin(PGI2) by normal human vascuiar endothelial cell (VEC). Methods Radioimmunoassay(RIA) was used to assess the amount of ET-1 and PGI2 produced by VEC. VEC prollferation was assessed by tetrazolium(MTT) assay. Results Que increased the normal VEC prollferation at the concentration or 5, 2o, 4o, 8o, 1oompol/L and increased the production of PG12 and inhibits the release of ET by the normal VEC at the concentratiou or 5, 2o and 8ompol/L. Que at the concentration of 5, 2o and 8omol/L had no direct effect on morphology of the normal VEC. ConcIusion Que can stimulate the proliferation of VEC and inhibit tbe reIease of ET-1 and increase the formation of PGI2. The data suggest that Que might be beneficial for the prevention and treatment of vascular endothelial injury-related cardiovascular diseases, such as atherosclerosls and thromboembolism diseases.

Regulation of opticin on bioactivity of retinal vascular endothelial cells cultured in collagen

AIM:To investigate the effects of collagen and opticin on the bioactivity of human retinal vascular endothelial cells(hR VECs),and explore its regulations by integrins and RhoA/ROCK1 signal pathway.METHODS:h RVECs were cultured in collagen and treated by opticin,and cell-based bioactivity assays of cell proliferation,migration,and adhesion were performed.The expression of integrinα2,integrinβ1,Rho A and ROCK1 were examined with real-time PCR and Western blotting.RESULTS:Collagen could promote cell viability of proliferation and migration(all P<0.05),and enhance the m RNA expression of integrinα2,integrinβ1,Rho A and ROCK1(all P<0.05).Opticin could inhibit proliferation and migration ability of hR VECs cultured in collagen,and reduce the mR NA expression of integrinα2,integrinβ1,RhoA and ROCK1(all P<0.05).CONCLUSION:Collagen and opticin can affect bioactivity of hR VECs,which may be regulated byα2-,β1-integrins and RhoA/ROCK1 signal pathway.

Claudin-1 Leads to Strong Formation of Tight Junction in Cultured Mouse Lung Microvascular Endothelial Cells

We aimed to examine paracellular barrier function in cultured mouse lung microvascular endothelial cells (LMECs). The transcellular resistance of LMEC monolayers yielded an electrical resistance of approximately 19 Ω × cm2 at days 6 - 7 in culture when the cells reached confluence, and paracellular permeable clearance of sodium fluorescein was the lowest on day 6 in culture, suggesting the formation of tight junctions (TJs) in cultured LMECs. Moreover, the expression of TJ-associated proteins, occludin, claudin-1 and -4 and zonula occludents 1 (ZO-1) was detected in LMECs at day 6 in culture. However, mRNAs of occludin, claudin-1 and -4 and ZO-1 were already expressed on day 1 after culture, and large variations were absent in the mRNA levels of occludin, claudin-4 and ZO-1 between days 1 and 7 in culture, when the level of each mRNA on day 1 in culture was used as a basal level. However, the claudin-1 mRNA level gradually increased up to approximately 7-fold on day 7 in culture over the basal level. These results indicate that the drastic increase in the mRNA expression level of claudin-1 leads to the strong formation of TJs.

Multifaceted roles of lymphatic and blood endothelial cells in the tumor microenvironment of hepatocellular carcinoma:A comprehensive review

The tumor microenvironment is a complex network of cells,extracellular matrix,and signaling molecules that plays a critical role in tumor progression and metastasis.Lymphatic and blood vessels are major routes for solid tumor metastasis and essential parts of tumor drainage conduits.However,recent studies have shown that lymphatic endothelial cells(LECs)and blood endothelial cells(BECs)also play multifaceted roles in the tumor microenvironment beyond their structural functions,particularly in hepatocellular carcinoma(HCC).This comprehensive review summarizes the diverse roles played by LECs and BECs in HCC,including their involvement in angiogenesis,immune modulation,lymphangiogenesis,and metastasis.By providing a detailed account of the complex interplay between LECs,BECs,and tumor cells,this review aims to shed light on future research directions regarding the immune regulatory function of LECs and potential therapeutic targets for HCC.