骨髓间充质干细胞移植对大鼠低氧性肺动脉高压的影响

目的：探讨骨髓间充质干细胞（MSCs）移植对大鼠低氧性肺动脉高压（HPH）的影响。方法：体外分离、培养、鉴定SD大鼠骨髓MSCs、绿色荧光蛋白腺病毒标记MSCs细胞。将健康雄性SD大鼠随机分为4组：正常对照组（NC组）8只、低氧性肺动脉高压组（HPH组）8只,低氧性肺动脉高压同时骨髓间充质干细胞移植组（MSCs组）24只,低氧性肺动脉高压同时携带血管内皮生长因子（VEGF）的MSCs移植组（VEGF＋MSCs组）24只。采用常压间歇低氧法建立大鼠肺动脉高压模型,干细胞转染并行干细胞移植。观察大鼠平均肺动脉压力（mPAP）,计算右心室肥厚指数（RVHI）,显微镜下观察各组大鼠肺小动脉形态结构改变,并在荧光显微镜下观察干细胞移植7d,14d,28d时腺病毒转染荧光标记的MSCs在肺小动脉分布及表现。结果：NC组28d时mPAP（mmHg）为15．5±1．5,而HPH组、MSCs组及MSCs＋VEGF组分另q为26．1±1．9、21．6±2．7及20．1±2．9,均明显高于NC组（P〈0．01）,但MSCs组及MSCs＋VEGF组较HPH组明显下降（P〈0．01）,MSCs组与MSCs＋VEGF组无明显差别。NC组28d时RVHI为0．28±0．02,而HPH组、MSCs组及MSCs＋VEGF组RVHI分别为0．43±0．07、0．34±0．03及0．35±0．01,均明显高于NC组（P〈0．01）,但MSCs组及MSCs＋VEGF组较HPH组明显下降（P〈0．05）,MSCs组与MSCs＋VEGF组无明显差别。HPH组28d时,肺小动脉管壁明显增厚,管腔明显狭窄、闭塞,内皮细胞不完整,而MSCs组血管壁较HPH组变薄,管腔通畅,内皮细胞完整性改善,MSC8组及MSCs＋VEGF组的表现改变不明显。结论：骨髓间充质干细胞移植可改善肺小动脉血管重塑,从而部分逆转HPH的进程;而将VEGF与MSCs联合移植并未提高单纯MSCs移植的作用。     

Surface expression of CXCR4 in unrestricted somatic stem cells and its regulation by growth factors

Umbilical cord blood‐derived USSCs (unrestricted somatic stem cells) have recently been considered as a potential source for stem cell therapy and transplantation due to their characteristics such as easy accessibility, low immunogenicity, self‐renewing and multilineage differentiation potential. Stem cell homing is a key factor in successful transplantation, which is regulated by CXCR4 in stem cells. In this study, we evaluated the expression of CXCR4 in USSCs different passages. Moreover, the effect of VEGF (vascular endothelial growth factor) and IGF‐1 (insulin‐like growth factor 1) on its expression was assessed. It was shown that the expression of CXCR4 in USSCs decreased with the increase in passage number. It was also revealed that VEGF increased surface expression and mRNA level of CXCR4 in USSCs, while IGF‐1 decreased its expression. When VEGF and IGF‐1 were administered simultaneously, CXCR4 expression was increased, but the expression level was less than VEGF alone. Finally, it was shown that over‐expression of CXCR4 enhanced the migratory capacity of USSCs. The increase of CXCR4 expression, here caused by VEGF in USSCs, can improve the efficacy of stem cell therapy and transplantation after long‐term culture of stem cells before clinical use.     

Mesenchymal stem cells stimulate angiogenesis in a murine xenograft model of A549 human adenocarcinoma through an LPA1 receptor-dependent mechanism

Carcinoma-associated fibroblasts play a key role in tumorigenesis and metastasis by providing a tumor-supportive microenvironment. In the present study, we demonstrate that conditioned medium from A549 human lung adenocarcinoma cells induces differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) to carcinoma-associated fibroblasts expressing α-smooth muscle actin, vascular endothelial growth factor, and stromal cell-derived factor-1. A549 conditioned medium-induced differentiation of hASCs to carcinoma-associated fibroblasts was completely abrogated by treatment of hASCs with Ki16425, a lysophosphatidic acid receptor antagonist, or knockdown of lysophosphatidic acid receptor 1 (LPA₁) expression in hASCs with small interfering RNA or lentiviral short hairpin RNA. Using a murine xenograft transplantation model of A549 cells, we showed that co-transplantation of hASCs with A549 cells stimulated growth of A549 xenograft tumor, angiogenesis, and differentiation of hASCs to carcinoma-associated fibroblasts in vivo. Knockdown of LPA₁ expression in hASCs abrogated hASCs-stimulated growth of A549 xenograft tumor, angiogenesis, and differentiation of hASCs to carcinoma-associated fibroblasts. Moreover, A549 conditioned medium-treated hASCs stimulated tube formation of human umbilical vein endothelial cells by LPA₁-dependent secretion of vascular endothelial growth factor. These results suggest that A549 cells induce in vivo differentiation of hASCs to carcinoma-associated fibroblasts, which play a key role in tumor angiogenesis within tumor microenvironment, through an LPA-LPA₁-mediated paracrine mechanism.     

Laminin-511 expression is associated with the functionality of feeder cells in human embryonic stem cell culture

Fibroblast feeder cells play an important role in supporting the derivation and long term culture of undifferentiated, pluripotent human embryonic stem cells (hESCs). The feeder cells secrete various growth factors and extracellular matrix (ECM) proteins into extracellular milieu. However, the roles of the feeder cell-secreted factors are largely unclear. Animal feeder cells and use of animal serum also make current feeder cell culture conditions unsuitable for derivation of clinical grade hESCs. We established xeno-free feeder cell lines using human serum (HS) and studied their function in hESC culture. While human foreskin fibroblast (hFF) feeder cells were clearly hESC supportive, none of the established xeno-free human dermal fibroblast (hDF) feeder cells were able to maintain undifferentiated hESC growth. The two fibroblast types were compared for their ECM protein synthesis, integrin receptor expression profiles and key growth factor secretion. We show that hESC supportive feeder cells produce laminin-511 and express laminin-binding integrins α3ß1, α6ß1 and α7ß1. These results indicate specific laminin isoforms and integrins in maintenance of hESC pluripotency in feeder-dependent cultures. In addition, several genes with a known or possible role for hESC pluripotency were differentially expressed in distinct feeder cells.     

Expressions of pathologic markers in PRP based chondrogenic differentiation of human adipose derived stem cells

Background

Optimization of the differentiation medium through using autologous factors such as PRP is of great consideration, but due to the complex, variable and undefined composition of PRP on one hand and lack of control over the absolute regulatory mechanisms in in vitro conditions or disrupted and different mechanisms in diseased tissue microenvironments in in vivo conditions on the other hand, it is complicated and rather unpredictable to get the desired effects of PRP making it inevitable to monitor the possible pathologic or undesired differentiation pathways and therapeutic effects of PRP. Therefore, in this study the probable potential of PRP on inducing calcification, inflammation and angiogenesis in chondrogenically-differentiated cells was investigated.

Regulation of adult neural precursor cell migration

Adult neural precursor cells (NPCs) are predominantly located in the subventricular zone (SVZ) of the lateral ventricles or in the subgranular zone of the dentate gyrus. These NPCs produce neuroblasts that normally migrate and integrate into the olfactory bulb and hippocampus, respectively. Following CNS damage due to disease or injury, NPCs can also migrate to the site of damage. Enhancement of NPC migration to sites of neural damage may increase their potential for repair but requires an understanding of processes that regulate basal and injury-induced migration so we can harness this potential. This review highlights the extrinsic factors and major intrinsic signalling pathways that regulate endogenous basal NPC migration to the olfactory bulb and the role of inflammatory mediators and chemokines in disease and injury-induced NPC migration.     

Protein profiling and angiogenic effect of hypoxia-cultured human umbilical cord blood-derived mesenchymal stem cells in hindlimb ischemia

The aim of the present study was to investigate protein profiles of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) cultured in normoxic (21% O₂) and hypoxic (1% O₂) conditions, and evaluate oxygenation effects on angiogenesis in an ischemic hindlimb mouse model using a modified ischemic scoring system. Hypoxic conditions did not change the expression of phenotypic markers and increased adipogenesis and chondrogenesis. Epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), TGF-β RII, and vascular endothelial growth factor (VEGF) were upregulated in the conditioned medium of hypoxic hUCB-MSCs, which are commonly related to angiogenesis and proliferation of biological processes by Gene Ontology. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, significant enrichment of the phosphorylation of abelson murine leukemia viral oncogene homolog 1 (ABL1) (Phospho-Tyr204) and B-cell lymphoma-extra large (BCL-XL) (Phospho-Thr47) as anti-apoptotic pathways was observed in hypoxic hUCB-MSCs. Furthermore, hypoxic conditions induced proliferation and migration, and reduced apoptosis of hUCB-MSCs in vitro. Based on the results of protein antibody array, we evaluated the angiogenic effects of injecting normoxic or hypoxic hUCB-MSCs (1 × 10⁶) into the ischemic hindlimb muscles of mice. Ischemic scores and capillary generation were significantly greater in the hypoxic hUCB-MSC injection group than in the normoxic hUCB-MSC group. Our findings demonstrate that culturing hUCB-MSCs in hypoxic conditions not only significantly enriches phosphorylation in the anti-apoptosis pathway and enhances the secretion of several angiogenic proteins from cells, but also alleviates ischemic injury of hindlimb of mice.     

Deconstructing breast cancer cell biology and the mechanisms of multidrug resistance

Cancer complexity constantly challenges the way that clinicians manage breast cancer therapy. Tumor heterogeneity and intratumoral stroma characteristics allow cells with different phenotypes and deregulated apoptotic, proliferative and migration abilities to co-exist contributing to a disappointing therapeutic response. While new approaches are being associated with conventional chemotherapy, such as hormonal therapy or target monoclonal antibodies, recurrence and metastasization are still observed. Membrane transporters are the cell's first line of contact with anticancer drugs having a major role in multidrug resistance events. This structural-based activity enables the cell to be drug-resistant by decreasing drug intracellular concentration through an efflux-transport mechanism, mainly associated with overexpression of ATP-binding cassette (ABC) proteins. This review focuses on some of the important structural and biological properties of the malignant cell and tumor microenvironment, addressing the role of the membrane ABC transporters in therapeutic outcomes, and highlighting related molecular pathways that may represent meaningful target therapies.     

Chitosan stabilizes platelet growth factors and modulates stem cell differentiation toward tissue regeneration

The idea of using chitosan as a functional delivery aid to support simultaneously PRP, stem cells and growth factors (GF) is associated with the intention to use morphogenic biomaterials to modulate the natural healing sequence in bone and other tissues. For example, chitosan–chondroitin sulfate loaded with platelet lysate was included in a poly(d,l-lactate) foam that was then seeded with human adipose-derived stem cells and cultured in vitro under osteogenic stimulus: the platelet lysate provided to the bone tissue the most suitable assortment of GF which induces the osteogenic differentiation of the mesenchymal stem cells. PDGF, FGF, IGF and TGF-β were protagonists in the repair of callus fractures. The release of GF from the composites of chitosan–PRP and either nano-hydroxyapatite or tricalcium phosphate was highly beneficial for enhancing MSC proliferation and differentiation, thus qualifying chitosan as an excellent vehicle. A number of biochemical characteristics of chitosan exert synergism with stem cells in the regeneration of soft tissues.     

GPI/AMF inhibition blocks the development of the metastatic phenotype of mature multi-cellular tumor spheroids

Epithelial–mesenchymal transition (EMT) and cellular invasiveness are two pivotal processes for the development of metastatic tumor phenotypes. The metastatic profile of non-metastatic MCF-7 cells growing as multi-cellular tumor microspheroids (MCTSs) was analyzed by determining the contents of the EMT, invasive and migratory proteins, as well as their migration and invasiveness potential and capacity to secrete active cytokines such as the glucose phosphate isomerase/AMF (GPI/AMF). As for the control, the same analysis was also performed in MCF-7 and MDA-MB-231 (highly metastatic, MDA) monolayer cells, and in stage IIIB and IV human metastatic breast biopsies. The proliferative cell layers (PRL) of mature MCF-7 MCTSs, MDA monolayer cells and metastatic biopsies exhibited increased cellular contents (2–15 times) of EMT (β-catenin, SNAIL), migratory (vimentin, cytokeratin, and fibronectin) and invasive (MMP-1, VEGF) proteins versus MCF-7 monolayer cells, quiescent cell layers of mature MCF-7 MCTS and non-metastatic breast biopsies. The increase in metastatic proteins correlated with substantially elevated cellular abilities for migration (18-times) and invasiveness (13-times) and with the higher level (6-times) of the cytokine GPI/AMF in the extracellular medium of PRL, as compared to MCF-7 monolayer cells. Interestingly, the addition of the GPI/AMF inhibitors erythrose-4-phosphate or 6-phosphogluconate at micromolar doses significantly decreased its extracellular activity (> 80%), with a concomitant diminution in the metastatic protein content and migratory tumor cell capacity, and with no inhibitory effect on tumor lactate production or toxicity on 3T3 mouse fibroblasts. The present findings provide new insights into the discovery of metabolic inhibitors to be used as complementary therapy against metastatic and aggressive tumors.     

New facets of matrix metalloproteinases MMP-2 and MMP-9 as cell surface transducers: Outside-in signaling and relationship to tumor progression

This review focuses on matrix metalloproteinases (MMPs)-2 (gelatinase A) and -9 (gelatinase B), both of which are cancer-associated, secreted, zinc-dependent endopeptidases. Gelatinases cleave many different targets (extracellular matrix, cytokines, growth factors, chemokines and cytokine/growth factor receptors) that in turn regulate key signaling pathways in cell growth, migration, invasion, inflammation and angiogenesis. Interactions with cell surface integral membrane proteins (CD44, αVβ/αβ1/αβ2 integrins and Ku protein) can occur through the gelatinases' active site or hemopexin-like C-terminal domain. This review evaluates the recent literature on the non-enzymatic, signal transduction roles of surface-bound gelatinases and their subsequent effects on cell survival, migration and angiogenesis. Gelatinases have long been drug targets. The current status of gelatinase inhibitors as anticancer agents and their failure in the clinic is discussed in light of these new data on the gelatinases' roles as cell surface transducers — data that may lead to the design and development of novel, gelatinase-targeting inhibitors.     

Inhibition of surgical trauma-enhanced peritoneal dissemination of tumor cells by human catalase derivatives in mice

Surgical trauma, which is inevitably associated with the surgical removal of cancer, has been reported to accelerate tumor metastasis. The close association of reactive oxygen species with the trauma and tumor metastasis supports the possibility of using antioxidants for the inhibition of metastasis. To inhibit surgical trauma-enhanced peritoneal dissemination, human catalase (hCAT) derivatives, i.e., hCAT-nona-arginine peptide (hCAT-R9) and hCAT-albumin-binding peptide (hCAT-ABP), were designed to increase the retention time of the antioxidant enzyme in the abdominal cavity after intraperitoneal administration. Both ¹²⁵I-labeled derivatives showed significantly prolonged retention in the cavity compared to ¹²⁵I-hCAT. Cauterization of the cecum of mice with a hot iron, an experimental model of surgical trauma, induced abdominal adhesions. In addition, cauterization followed by colon26 tumor cell inoculation increased lipid peroxidation in the cecum and mRNA expression of molecules associated with tissue repair/adhesion and inflammation in the peritoneum. hCAT derivatives significantly suppressed the increased mRNA expression. The cauterization also increased the number of tumor cells in the abdominal organs, and the number was significantly reduced by hCAT-R9 or hCAT-ABP. These results indicate that hCAT-R9 and hCAT-ABP, both of which have a long retention time in the peritoneal cavity, can be effective at inhibiting surgery-induced peritoneal metastasis.     

The pro-tumorigenic effects of metabolic alterations in glioblastoma including brain tumor initiating cells

De-regulated cellular energetics is an emerging hallmark of cancer with alterations to glycolysis, oxidative phosphorylation, the pentose phosphate pathway, lipid oxidation and synthesis and amino acid metabolism. Understanding and targeting of metabolic reprogramming in cancers may yield new treatment options, but metabolic heterogeneity and plasticity complicate this strategy. One highly heterogeneous cancer for which current treatments ultimately fail is the deadly brain tumor glioblastoma. Therapeutic resistance, within glioblastoma and other solid tumors, is thought to be linked to subsets of tumor initiating cells, also known as cancer stem cells. Recent profiling of glioblastoma and brain tumor initiating cells reveals changes in metabolism, as compiled here, that may be more broadly applicable. We will summarize the profound role for metabolism in tumor progression and therapeutic resistance and discuss current approaches to target glioma metabolism to improve standard of care.     

Deregulated signalling networks in human brain tumours

Despite the variety of modern therapies against human brain cancer, in its most aggressive form of glioblastoma multiforme (GBM) it is a still deadly disease with a median survival of approximately 1 year. Over the past 2 decades, molecular profiling of low- and high-grade malignant brain tumours has led to the identification and molecular characterisation of mechanisms leading to brain cancer development, maintenance and progression. Genetic alterations occurring during gliomagenesis lead to uncontrolled tumour growth stimulated by deregulated signal transduction pathways. The characterisation of hyperactivated signalling pathways has identified many potential molecular targets for therapeutic interference in human gliomas. Overexpressed or mutated and constitutively active kinases are attractive targets for low-molecular-weight inhibitors. Although the first attempts with mono-therapy using a single targeted kinase inhibitor were not satisfactory, recent studies based on the simultaneous targeting of several core hyperactivated pathways show great promise for the development of novel therapeutic approaches. This review focuses on genetic alterations leading to the activation of key deregulated pathways in human gliomas.