Lamei Cheng

LFA-1 and VLA-4 involved in human high proliferative potentialendothelial progenitor cells homing to ischemic tissue

Cumulative evidences have revealed that endothelial progenitor cell (EPC) transplantation can promote the neovascularization in ischemic tissue, but the mechanism of EPCs homing to the site of ischemia is poorly understood. In this study, to investigate the mechanism of human umbilical cord blood-derived high proliferative potential-endothelial progenitor cells (HPP-EPCs) homing to ischemic tissue we evaluated the expression of lymphocyte function-associated antigen-1 (LFA-1, or CD11a/CD18) and very late antigen-4 (VLA-4, or CD49d/CD29) in EPCs and the changes of expression level of their ligands, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), in ischemic tissue and performed the adhesion and migration assays to analyze the interaction between the receptors and ligands. Furthermore, we studied the roles of LFA-1 and VLA-4 in EPC homing in an ischemic model of mice. The results show that LFA-1 andVLA-4 were expressed in HPPEPCs and ICAM-1 and VCAM-1 were expressed in vessel endothelium in ischemic tissues. The pre-incubation of HPP-EPCs with neutralizing antibodies against CD11a or CD49d reduced adhesion and migration of HPP-EPCs in vitro and reduced recovery of hind-limb blood flow, capillary density and incorporation of HPP-EPC into ischemic tissues in vivo. Furthermore, the pre-incubation of HPP-EPCs with the combination of CD11a and CD49d antibodies led to synergistically negative effects on adhesion and transmigration of HPP-EPCs in vitro, and on the homing of HPP-EPCs to ischemic tissue and on neovascularization capacity in vivo. These results indicate that LFA-1 andVLA-4 are involved in HPP-EPC homing to ischemic tissues.

Angiogenic potential difference between two types of endothelial progenitor cells from human umbilical cord blood

The hierarchy of endothelial progenitor cells (EPCs) in human umbilical cord blood has been disclosed. In this study we compare, for the first time, the angiogenic potential difference between two types of EPCs. We cultured mononuclear cells (MNCs) isolated from human umbilical cord blood using endothelial cell‐conditioned medium and obtained two types of EPCs, referred to as circulating angiogenic cells (CACs) and high proliferative potential endothelial progenitor cells (HPP‐EPCs). Both types of cells possess characteristics of EPCs, including expressing CD31, VE‐cadherin, KDR and von Willebrand factor, uptake of Ac‐LDL and binding to lectin. However, unlike CACs, which express CD14 but not CD133, HPP‐EPCs express CD133 but not CD14. Also, unlike CACs, HPP‐EPCs display stronger proliferation and clonogenic potential in vitro and show stronger ability to promote vascular growth in the hind‐limb model of ischemia in mice (BALB/C‐nu) in vivo.

Effect of oxidized low-density lipoprotein on survival and function of endothelial progenitor cell mediated by p38 signal pathway.

This study was designed to investigate whether oxidized low-density lipoprotein (oxLDL) affects the survival and activity of endothelial progenitor cell (EPC) mediated by p38 mitogen-activated protein kinase (MAPK). EPCs were isolated from human peripheral blood in endothelial cell growth medium-2. Incubation with oxLDL at 100 μg/mL decreased EPC number. Treated with oxLDL resulted in increase of EPC apoptosis and in decrease of EPC proliferation. Treatment with oxLDL resulted in a significantly reduced migratory rate of EPCs and reduced adhesion to fibronectin. Treatment with oxLDL impaired the in vitro angiogenesis ability of EPCs. However, all the detrimental effects on EPC were attenuated by pretreatment of EPCs with SB203580, an inhibitor of the p38 MAPK. In addition, the inhibition of the p38-kinase by SB203580 also significantly improved basal number and functions of EPCs. Western blot analysis revealed that oxLDL induced dose- and time-dependent activation of the p38 MAPK. These results demonstrated that p38 MAPK plays a critical role in regulating the number and functions of EPCs in vitro. SB203580 can improve the number and functions of EPCs under basal conditions and prevent the negative effects of oxLDL on the number and functions of EPCs and may be useful to improve the number and function of EPCs for potential cell therapy.

Characterization and comparison of embryonic stem cell-derived KDR + cells with endothelial cells

Growing interest in utilizing endothelial cells (ECs) for therapeutic purposes has led to the exploration of human embryonic stem cells (hESCs) as a potential source for endothelial progenitors. In this study, ECs were induced from hESC lines and their biological characteristics were analyzed and compared with both cord blood endothelial progenitor cells (CBEPCs) and human umbilical vein endothelial cells (HUVECs) in vitro. The results showed that isolated embryonic KDR+ cells (EC-KDR+) display characteristics that were similar to CBEPCs and HUVECs. EC-KDR+, CBEPCs and HUVECs all expressed CD31 and CD144, incorporated DiI-Ac-LDL, bound UEA1 lectin, and were able to form tube-like structures on Matrigel. Compared with CBEPCs and HUVECs, the expression level of endothelial progenitor cell markers such as CD133 and KDR in EC-KDR+ was significantly higher, while the mature endothelial marker vWF was lowly expressed in EC-KDR+. In summary, the study showed that EC-KDR+ are primitive endothelial-like progenitors and might be a potential source for therapeutic vascular regeneration and tissue engineering.

Effects of an endothelial cell-conditioned medium on the hematopoietic and endothelial differentiation of embryonic stem cells

We have examined the effect of mouse bone marrow endothelial cell‐conditioned medium (mEC‐CM) on hematopoietic and endothelial differentiation of mouse embryonic stem cells (mESCs). mEC‐CM can efficiently promote the differentiation of mESCs into Flk+ cells and hematopoietic colony‐forming cells. mEC‐CM proved to be as potent as a cytokine cocktail comprised of VEGF, bFGF, IGF and EGF. After inducing mESCs with mEC‐CM, cobblestone‐like cells were mechanically selected and identified which had the ability to incorporate DiI‐Ac‐LDL. DiI‐Ac‐LDL‐positive cells were endothelial‐like cells due to their expression of CD31 and Flk1, ability to bind to UEA1 and capacity to form capillary‐like tube structures on matrigel. In conclusion, mEC‐CM can efficiently promote the differentiation of mESCs into endothelial cells and hematopoietic colony‐forming cells. The differentiated endothelial‐like cells can be isolated by using DiI‐Ac‐LDL labeling and mechanical selection.

Expression of polypeptide GalNAc‐transferases in hematopoietic stem/progenitor cells

The members of the UDP‐GalNAc:polypeptide N‐acetylgalactosaminyltransferase (pp‐GalNAc‐T) family, which transfer GalNAc to polypeptide serine and threonine residues, initiate mucin‐type O‐linked glycosylation. There are at least 13 functionally characterized members of this family in humans, but no studies have been reported of pp‐GalNAc‐T isoforms in hematopoietic cells. We isolated and purified CD34+ hematopoietic cells from adult bone marrow by magnetic cell sorting and induced them to differentiate into megakaryocytic lineage cells using an optimal combination of hematopoietic growth factors in serum‐free liquid medium. RT‐PCR revealed that CD34+ cells expressed pp‐GalNAc‐T1, T2, T3, T4, T6, T7, T10, T11 and T14, but not pp‐GalNAc‐T8, T9, T12 and T13. The megakaryocytic lineage cells showed significant increases in the expression of pp‐GalNAc‐T3, T8, T9, T10 and T13, but pp‐GalNAc‐T11 and T14 became undetectable. In summary, many pp‐GalNAc‐T isoforms were expressed in CD34+ cells but the expression pattern changed during differentiation into megakaryocytes. The expression patterns of pp‐GalNAc‐Ts may be necessary to ensure proper O‐glycosylation of mucin‐type proteins expressed in CD34+ and megakaryocytic cells.

Several questions in differentiation of human embryonic stem cells to endothelial cells

There are accumulating knowledge and technologies in induction and identification of human embryonic stem cells(hESCs) derived endothelial cells. However, the variation of the results in different laboratories is large, and the reproducibility of the experiments is not reliable. There are three probable reason: 1. The differences of hESC lines; 2. The differences of culture condition; 3. The differences of sorting markers that have been used to purify endothelial cell population. In this study, 5 normal hESC lines from Asian race were established and employed to differentiate into endothelial cells. After 9 days spontaneous differentiation of embryoid bodies (EBs), the percentage of KDR (17-45%), VE-cadherin(0.2-0.9%) and CD34(1.7-2.8%) positive cells showed significant differences compared with previously reported results; but did not show significant differences among 5 cell lines. We isolated KDR+ cells from 9-day-old EBs by immunomagnetic beads sorting, these cells were plated in EGM-2 medium supplemented with vascular endothelial growth factor (VEGF). After 7 days culture, these cells expressed KDR, VE-cadherin, PECAM1, UEA1 but not mature endothelial cell protein vWF; and were capable of taking up DiI-Ac-LDL and forming capillary-like tube structure on matrigel, thus these cells are primitive endothelial-like cells. KDR derived endothelial-like cells showed the similar abilities of proliferation and capillary-like structure formation when compared with endothelial progenitor cells derived from cord blood. The results suggest that the KDR+ cells-derived cells are a kind of primitive endothelial-like precursors.

SPARC support the expansion of cord blood stem cells in vitro

SPARC (secreted protein, acidic and rich in cysteine) is a multifunctional protein that is associated with morphogenesis, remodeling, cellular migration, and proliferation. SPARC is abundantly expressed in osteoblasts and endothelial cells which contribute to the creation of HSC(hematopoietic stem cells) niches within the bone marrow, however, its function in hematopoiesis is not clear. In this study, we explored the roles and molecular profile of SPARC in regulating hematopoiesis. Exogenous SPARC could markedly promote the formation of BFU-E (erythroid burst-forming units) from cord blood CD34+ cells in a dose-dependent manner, but not CFUGM (colony-forming unit-granulocyte macrophage) and CFU-GEMM (colony-forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte). CD34+ cells were cultured in IMDM containing 10% FBS supplemented with cytokine combination (SCF, TPO, FL and G-CSF) with or without SPARC for 7 days, the total number of nucleated cells were expanded 163.1and 105.6-fold, and CD34+ cells were expanded 46.4and 26. 6-fold, respectively; SPARC also significantly promoted the expansion of CD34+-derived hematopoietic progenitors, BFU-E was expanded 19.6and 9.4-fold with or without SPARC respectively. To understand the possible molecular mechanisms underlying effects of SPARC on hematopoietic cells in vitro, gene expression profile was analyzed using Atlas human cDNA expression array after cord blood CD34+ cells were cultured in medium mentioned above for 24 h. The results showed that the expression level of 66 genes were significantly up-regulated, such as those related to the self-renewal and proliferation (c-Myc, C-Rel, SRC1£¬ErbB-3, PGF), hematopoietic differentiation (TGF-beta3, CD40, GATA3, MAL, IL10, IL13), cell adhesion and migration (CXCR4 CD44 HOX4£¬integrin-beta 3), anti-apoptosis signaling pathway (STAT6, caspase 9), and some transcription factors (TYK2, MAP2K6, PAX5, SP1); while the expression of only 3 gene transcripts (IL7R, IFNGR2,GAP43) were significantly down-regulated. These results demonstrate that SPARC could regulate the expression of various genes related to proliferation, differentiation and anti-apoptosis and untimatly resulted in the promotion of proliferation and definitive differentiation, and inhibition of apoptosis of cord blood CD34+ cells. Our results also suggest that SPARC, which is expressed abundantly in bone marrow microenvironment, may play a very important role in hematopoietic hemeostasis in vivo.

IGF-1 reduces the apoptosis of endothelial progenitor cells induced by oxidized low-density lipoprotein by the suppressing caspasse-3 activity

IGF-1 reduces the apoptosis of endothelial progenitor cells induced by oxidized low-density lipoprotein by the suppressing caspasse-3 activity

Angiotensin II induced the apoptosis of the endothelial progenitor cells by the caspase-3

Endothelial regeneration play an important role in restoring vascular tone in respone to vascular injury. And the increased level of angiotensin II (Ang II) is an important risk factor in endothelial injury, which has been considered to play a key role in the initiation and development of atherosclerosis. Endothelial progenitor cells (EPCs) have been shown to enhance the formation of a new endothelim and reendotheliazation. But in previous study, Ang II affects the apoptosis of the EPCs. However, the precise mechanism is still unclear. Therefore, we focused in this study to understand whether Ang II can induce the eNOS (and caspase-3 activity in the EPCs. EPCs were isolated from peripheral blood and characterized. The EPCs were cultured then were treated with Ang II for 48 h. The apoptosis of the endothelial cells was checked by the flow cytometry and, while caspase-3 activity and eNOS was measured simultaneously. After the EPC were treated, the mean of apoptotic rates and caspase-3 enzyme activity and eNOS significantly decreased. Ang II marked inducd a cell cycle at G0/G1 phase and cell apoptosis. and the Angiotension II type1 receptor mRNA is upregulation. therefore Ang II stimulated caspase-3 enzyme leading to apoptosis of the EPCs.