Exosomes Derived from Hypoxic Leukemia Cells Enhance Tube Formation in Endothelial Cells

Hypoxia plays an important role during the evolution of cancer cells and their microenvironment. Emerging evidence suggests communication between cancer cells and their microenvironment occurs via exosomes. This study aimed to clarify whether hypoxia affects angiogenic function through exosomes secreted from leukemia cells. We used the human leukemia cell line K562 for exosome-generating cells and human umbilical vein endothelial cells (HUVECs) for exosome target cells. Exosomes derived from K562 cells cultured under normoxic (20%) or hypoxic (1%) conditions for 24 h were isolated and quantitated by nanoparticle tracking analysis. These exosomes were then cocultured with HUVECs to evaluate angiogenic activity. The exosomes secreted from K562 cells in hypoxic conditions significantly enhanced tube formation by HUVECs compared with exosomes produced in normoxic conditions. Using a TaqMan low-density miRNA array, we found a subset of miRNAs, including miR-210, were significantly increased in exosomes secreted from hypoxic K562 cells. We demonstrated that cancer cells and their exosomes have altered miRNA profiles under hypoxic conditions. Although exosomes contain various molecular constituents such as proteins and mRNAs, altered exosomal compartments under hypoxic conditions, including miR-210, affected the behavior of endothelial cells. Our results suggest that exosomal miRNA derived from cancer cells under hypoxic conditions may partly affect angiogenic activity in endothelial cells.     

Prolonged hypoxic culture and trypsinization increase the pro-angiogenic potential of human adipose tissue-derived stem cells

Background aimsTransplantation of mesenchymal stromal cells (MSC), including adipose tissue-derived stem cells (ASC), is a promising option in the treatment of vascular disease. Short-term hypoxic culture of MSC augments secretion of anti-apoptotic and angiogenic cytokines. We hypothesized that prolonged hypoxic (1% and 5% oxygen) culture and trypsinization would augment ASC expression of anti-apoptotic and angiogenic cytokines and increase the angiogenic potential of ASC-conditioned media.MethodsThe effects of prolonged hypoxic culture on growth and pro-angiogenic properties were investigated using human ASC cultured at 1%, 5% and 21% oxygen. The effect of trypsinization on the expression of pro-angiogenic genes was also determined.ResultsTrypsinization induced up-regulation of the vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) genes independent of oxygen concentration. The expression of VEGF and IGF-1 was up-regulated in ASC cultured at 1% oxygen for 13 days compared with 4 days. The VEGF concentration in ASC-conditioned media was higher after prolonged hypoxic culture compared with short-term culture, while the IGF-1 and chemokine (CXC motif) ligand 12 (CXCL12) concentrations were unchanged. The VEGF receptor blocker SU5416 abolished angiogenesis in a cultured rat aortic ring model. Media from cells exposed to hypoxia increased angiogenesis, an effect that was dependent on factors other than just the VEGF concentration in the added media.ConclusionsOptimization of the angiogenic potential of stem cell-based therapy in the treatment of vascular disease is important. We have demonstrated that prolonged hypoxic culture and trypsinization augment the therapeutic angiogenic potential of ASC.     

Modulation of Angiogenic and Inflammatory Response in Glioblastoma by Hypoxia

Glioblastoma are rapidly proliferating brain tumors in which hypoxia is readily recognizable, as indicated by focal or extensive necrosis and vascular proliferation, two independent diagnostic criteria for glioblastoma. Gene expression profiling of glioblastoma revealed a gene expression signature associated with hypoxia-regulated genes. The correlated gene set emerging from unsupervised analysis comprised known hypoxia-inducible genes involved in angiogenesis and inflammation such as VEGF and BIRC3, respectively. The relationship between hypoxia-modulated angiogenic genes and inflammatory genes was associated with outcome in our cohort of glioblastoma patients treated within prospective clinical trials of combined chemoradiotherapy. The hypoxia regulation of several new genes comprised in this cluster including ZNF395, TNFAIP3, and TREM1 was experimentally confirmed in glioma cell lines and primary monocytes exposed to hypoxia in vitro. Interestingly, the cluster seems to characterize differential response of tumor cells, stromal cells and the macrophage/microglia compartment to hypoxic conditions. Most genes classically associated with the inflammatory compartment are part of the NF-kappaB signaling pathway including TNFAIP3 and BIRC3 that have been shown to be involved in resistance to chemotherapy.Our results associate hypoxia-driven tumor response with inflammation in glioblastoma, hence underlining the importance of tumor-host interaction involving the inflammatory compartment.     

Hypoxia induces osteogenic/angiogenic responses of bone marrow‐derived mesenchymal stromal cells seeded on bone‐derived scaffolds via ERK1/2 and p38 pathways

Osteogenesis and angiogenesis are tightly coupled processes during bone development and formation. It is thus well known that the enhancement of vascularization is of great importance in bone tissue engineering. As a potential approach for repairing bone defects, bone tissue constructs should therefore replicate the essential components in vivo microenvironments to promote cell osteogenic differentiation while at same time induce angiogenic response. In light of standpoint above, a combination of human bone‐derived scaffolds and BMSCs that subjected to hypoxia was used to mimic in vivo conditions. Also the underlying cellular/molecular regulation was fully investigated. The results showed that hypoxia (5–10% O₂) greatly enhanced the proliferation of BMSCs seeded in scaffolds, although the hypoxia (5% O₂)‐induced proliferative effect on BMSC cellular scaffolds was not apparent to those cultured in plates. However, such a kind of model was able to significantly induce the osteogenic/angiogenic responses of BMSCs as reflected by osteogenesis or angiogenesis‐related highly expressed genes or proteins, such as alkaline phosphatase, osteocalcin, hypoxia‐inducible factor‐1α and vascular endothelial growth factor. Moreover, ERK1/2 and/or p38 pathways were demonstrated to play essential roles in hypoxia‐induced osteogenic/angiogenic responses. Our results indicated that the combination of bone‐derived scaffolds, a material that has a three dimensional network structure, and hypoxia, an environment that replicates in vivo BMSCs hypoxic living conditions, may be a potential approach for creating functional tissue‐engineered bone. Biotechnol. Bioeng. 2013; 110: 1794–1804. © 2013 Wiley Periodicals, Inc.     

Hypoxic expression of NLRP3 and VEGF in cultured retinal pigment epithelial cells: contribution of P2Y<Subscript>2</Subscript> receptor signaling

Retinal hypoxia is a major condition of the chronic inflammatory disease age-related macular degeneration. Extracellular ATP is a danger signal which is known to activate the NLRP3 inflammasome in various cell systems. We investigated in cultured human retinal pigment epithelial (RPE) cells whether hypoxia alters the expression of inflammasome-associated genes and whether purinergic receptor signaling contributes to the hypoxic expression of key inflammatory (NLRP3) and angiogenic factor (VEGF) genes. Hypoxia and chemical hypoxia were induced by a 0.2%-O₂ atmosphere and addition of CoCl₂, respectively. Gene expression was determined with real-time RT-PCR. Cytosolic NLRP3 and (pro-) IL-1β levels, and the extracellular VEGF level, were evaluated with Western blot and ELISA analyses. Cell culture in 0.2% O₂ induced expression of NLRP3 and pro-IL-1β genes but not of the pro-IL-18 gene. Hypoxia also increased the cytosolic levels of NLRP3 and (pro-) IL-1β proteins. Inflammasome activation by lysosomal destabilization decreased the cell viability under hypoxic, but not control conditions. In addition to activation of IL-1 receptors, purinergic receptor signaling mediated by a pannexin-dependent release of ATP and a release of adenosine, and activation of P2Y₂ and adenosine A₁ receptors, was required for the full hypoxic expression of the NLRP3 gene. P2Y₂ (but not A₁) receptor signaling also contributed to the hypoxic expression and secretion of VEGF. The data indicate that hypoxia induces priming and activation of the NLRP3 inflammasome in cultured RPE cells. The hypoxic NLRP3 and VEGF gene expression and the secretion of VEGF are in part mediated by P2Y₂ receptor signaling.     

Hypoxia regulates VEGF expression and cellular proliferation by osteoblasts in vitro.

Numerous studies have demonstrated the critical role of angiogenesis for successful osteogenesis during endochondral ossification and fracture repair. Vascular endothelial growth factor (VEGF), a potent endothelial cell-specific cytokine, has been shown to be mitogenic and chemotactic for endothelial cells in vitro and angiogenic in many in vivo models. Based on previous work that (1) VEGF is up-regulated during membranous fracture healing, (2) the fracture site contains a hypoxic gradient, (3) VEGF is up-regulated in a variety of cells in response to hypoxia, and (4) VEGF is expressed by isolated osteoblasts in vitro stimulated by other fracture cytokines, the hypothesis that hypoxia may regulate the expression of VEGF by osteoblasts was formulated. This hypothesis was tested in a series of in vitro studies in which VEGF mRNA and protein expression was assessed after exposure of osteoblast-like cells to hypoxic stimuli. In addition, the effects of a hypoxic microenvironment on osteoblast proliferation and differentiation in vitro was analyzed. These results demonstrate that hypoxia does, indeed, regulate expression of VEGF in osteoblast-like cells in a dose-dependent fashion. In addition, it is demonstrated that hypoxia results in decreased cellular proliferation, decreased expression of proliferating cell nuclear antigen, and increased alkaline phosphatase (a marker of osteoblast differentiation). Taken together, these data suggest that osteoblasts, through the expression of VEGF, may be in part responsible for angiogenesis and the resultant increased blood flow to fractured bone segments. In addition, these data provide evidence that osteoblasts have oxygen-sensing mechanisms and that decreased oxygen tension can regulate gene expression, cellular proliferation, and cellular differentiation.     

Hypoxic stress enhances osteoclast differentiation via increasing IGF2 production by non-osteoclastic cells

Development of bone depends on a continuous supply of bone-degrading osteoclasts. Although several factors such as cytokines and integrins have been shown to be important for osteoclast recruitment, their mechanism of action is poorly understood. In this study, we demonstrated the enhancement of osteoclast formation by hypoxia and investigated the molecular mechanisms involved. Primary mouse bone marrow cells were cultured in normoxic and hypoxic conditions, and RNA was prepared from each group of cells. Total RNAs were applied to a DNA microarray analysis and then RT-PCR was performed to confirm the microarray data. The most interesting finding of our microarray analysis was upregulation of insulin-like growth factor 2 (IGF2) and stromal cell-derived factor 1 (SDF1) under hypoxic conditions. RT-PCR analysis revealed that IGF2 expression was markedly upregulated in the non-osteoclastic cells. The addition of exogenous IGF2 increased the number of osteoclastic TRAP-positive multinuclear cells formed under normoxic conditions, whereas the addition of exogenous SDF1 did not change osteoclast formation. These results suggest that the upregulation of IGF2 derived from non-osteoclastic cells might be a crucial factor for osteoclast differentiation.     

Upregulations of glucocorticoid-induced leucine zipper by hypoxia and glucocorticoid inhibit proinflammatory cytokines under hypoxic conditions in macrophages

Hypoxia and inflammation often develop concurrently in numerous diseases, and the influence of hypoxia on natural evolution of inflammatory responses is widely accepted. Glucocorticoid-induced leucine zipper (GILZ) is thought to be an important mediator of anti-inflammatory and immune-suppressive actions of glucocorticoid (GC). However, whether GILZ is involved in hypoxic response is still unclear. In this study, we investigated the effects of hypoxic exposure and/or the administration of dexamethasone (Dex), a synthetic GC on GILZ expression both in vitro and in vivo, and further explored the relationship between GILZ and proinflammatory cytokines IL-1β, IL-6, and TNF-α under normoxic and hypoxic conditions. We found that hypoxia not only remarkably upregulated the expression of GILZ, but also significantly enhanced Dex-induced expression of GILZ in macrophages and the spleen of rats. ERK activity is found involved in the upregulation of GILZ induced by hypoxia. Inhibiting the expression of GILZ in RAW264.7 cells using specific GILZ small interfering RNA led to a significant increase in mRNA production and protein secretion of IL-1β and IL-6 in hypoxia and abrogated the inhibitory effect of Dex on expression of IL-1β and IL-6 in hypoxia. We also found that adrenal hormones played pivotal roles in upregulation of GILZ expression in vivo. Altogether, data presented in this study suggest that GILZ has an important role not only in adjusting adaptive responses to hypoxia by negatively regulating the activation of macrophages and the expression of proinflammatory cytokines, but also in mediating the anti-inflammatory action of GC under hypoxic conditions.     

Morphologic and transcriptomic comparison of adipose- and bone-marrow-derived porcine stem cells cultured in alginate hydrogels

Advances in bioengineering, material chemistry, and developmental biology have led to the design of three-dimensional (3D) culture systems that better resemble the surrounding structure and chemistry of the in situ niches of cells in tissues. This study was designed to characterize and compare porcine adipose-derived stem cells (ADSC) and bone-marrow-derived stem cells (BMSC) induced to differentiate toward osteogenic and adipogenic lineages in vitro by using a 3D alginate hydrogel. The morphology and gene expression of the two cell populations during differentiation were analyzed. Both ADSC and BMSC showed morphological evidence of osteogenic and adipogenic differentiation. Expression patterns of genes characteristic of the onset of osteogenic differentiation (ALP, COL1A1, SPARC, SPP1) were low at the beginning of culture and generally increased during the period of differentiation up to 28 days in culture. Expression of genes associated with adipogenic differentiation (ACSL1, ADFP, ADIPOQ, CD36, DBI, DGAT2, PPARG, SCD) was consistently increased in ADSC cultured in alginate hydrogel relative to the start of differentiation. However, adipogenic gene expression of BMSC cultured in alginate hydrogel was more limited when compared with that of ADSC. Evaluation of cell numbers (via the MTT staining assay) suggested a greater viability of BMSC under osteogenic conditions in alginate hydrogels than under adipogenic conditions, whereas ADSC had greater viability under adipogenic conditions than under osteogenic conditions. This study thus provides an important initial evaluation of ADSC and BMSC seeded and differentiated toward the osteogenic and adipogenic cell lineages in a 3D alginate hydrogel in vitro.     

Increased Proliferation and Analysis of Differential Gene Expression in Human Wharton's Jelly-derived Mesenchymal Stromal Cells under Hypoxia

Multipotent mesenchymal stromal cells (MSCs) from Wharton''s jelly (WJ) of umbilical cord bear higher proliferation rate and self-renewal capacity than adult tissue-derived MSCs and are a primitive stromal cell population. Stem cell niche or physiological microenvironment plays a crucial role in maintenance of stem cell properties and oxygen concentration is an important component of the stem cell niche. Low oxygen tension or hypoxia is prevalent in the microenvironment of embryonic stem cells and many adult stem cells at early stages of development. Again, in vivo, MSCs are known to home specifically to hypoxic events following tissue injuries. Here we examined the effect of hypoxia on proliferation and in vitro differentiation potential of WJ-MSCs. Under hypoxia, WJ-MSCs exhibited improved proliferative potential while maintaining multi-lineage differentiation potential and surface marker expression. Hypoxic WJ-MSCs expressed higher mRNA levels of hypoxia inducible factors, notch receptors and notch downstream gene HES1. Gene expression profile of WJ-MSCs exposed to hypoxia and normoxia was compared and we identified a differential gene expression pattern where several stem cells markers and early mesodermal/endothelial genes such as DESMIN, CD34, ACTC were upregulated under hypoxia, suggesting that in vitro culturing of WJ-MSCs under hypoxic conditions leads to adoption of a mesodermal/endothelial fate. Thus, we demonstrate for the first time the effect of hypoxia on gene expression and growth kinetics of WJ-MSCs. Finally, although WJ-MSCs do not induce teratomas, under stressful and long-term culture conditions, MSCs can occasionally undergo transformation. Though there were no chromosomal abnormalities, certain transformation markers were upregulated in a few of the samples of WJ-MSCs under hypoxia.     

Hypoxic adipocytes induce macrophages to release inflammatory cytokines that render skeletal muscle cells insulin resistant

Adipose tissue hypoxia occurs early in obesity and is associated with increased tissue macrophages and systemic inflammation that impacts muscle insulin responsiveness. We investigated how hypoxia interacted with adipocyte-macrophage crosstalk and inflammatory cytokine release, using co-culture and conditioned media (CM). Murine primary adipocytes from lean or obese mice were cultured under normoxic (21% O₂) or hypoxic (1% O₂) conditions. RAW264.7 macrophages were incubated under normoxic or hypoxic conditions with or without adipocyte conditioned media. Macrophage and adipocyte-macrophage co-culture CM were also collected. We found hypoxia did not elicit direct cytokine release from macrophages. However, adipocyte CM or adipocyte co-culture, synergistically stimulated TNFα and MCP-1 release from macrophages that was not further impacted by hypoxia. Exposure of muscle cells to elevated cytokines led to reduced insulin and muscle stress/inflammatory signaling. We conclude hypoxia or obesity induces release of inflammatory TNFα and MCP-1 from mice primary adipocytes but the two environmental conditions do not synergize to worsen macrophage signal transduction or insulin responsiveness.     

Enhancement of differentiation efficiency of hESCs into vascular lineage cells in hypoxia via a paracrine mechanism

Hypoxia is one way of inducing differentiation due to the activation of the key regulatory factor, Hypoxia-inducible factor 1 alpha (HIF-1α). However, the action of HIF-1α on the differentiation of hESCs was unclear until now. To investigate the effect of hypoxia on the differentiation of hESCs, we compared the differentiation efficacy into vascular lineage cells under normoxic and hypoxic conditions. We observed HIF-1α expression and the related expression of pro-angiogenic factors VEGF, bFGF, Ang-1 and PDGF in hEBs cultured under hypoxic conditions. Along with this, differentiation efficacy into vascular lineage cells was improved under hypoxic conditions. When HIF-1α was blocked by echinomycin, both angiogenic factors and the differentiation efficacy were down-regulated, suggesting that the enhancement of differentiation efficacy was caused by intrinsic up-regulation of HIF-1α and these pro-angiogenic factors under hypoxic condition. This response might be primarily regulated by the HIF-1α signal pathway, and hypoxia might be the key to improving the differentiation of hESCs into vascular lineage cells. Therefore, this study demonstrated that microenvironmental changes (i.e., hypoxia) can improve differentiation efficacy of hESCs into a vascular lineage without exogenous factors via cell-intrinsic up-regulation of angiogenic factors. These facts will contribute to the regulation of stem cell fate.     

Hypoxia modulates cyclin and cytokine expression and inhibits peripheral mononuclear cell proliferation.

Previously, we found that hypoxia can deeply affect the production of cytokines in human peripheral mononuclear cells (PBMC). Here, we demonstrated that the cycle progression of hypoxic PBMC, cultured in the presence or not of a specific T cell activator such as phytohaemagglutinin (PHA), was delayed when compared with aerobic cultures. This delay was accompanied by a decrease of the expression of specific cyclins associated to cell cycle progression phases. Ribonuclease Protection Assay (RPA) studies reveal a decrease in the expression of cyclin A and B in PHA-stimulated PBMC kept for 40 hr under hypoxic condition (2% O(2)), when compared with aerobic cultures (20% O(2)). In concomitance, a decrease of cyclin D2 expression was present after 16 hr of hypoxic treatment. However, the decrease was transient and disappeared after 40 hr of hypoxic treatment. Furthermore, cyclin C expression was not affected by hypoxia. Hypoxia-induced cyclin modulation was accompanied by an increased synthesis of interleukin (IL)-2 and IL-4, analyzed by ELISA. By evaluating these results, it appears that hypoxia induces a growth suppressive state in mitogen-activated PBMC by inhibiting the synthesis of mitotic cyclins A and B. However hypoxic PBMC maintain their viability and capability of producing stimulatory cytokines, after mitogen treatment. This should be important in local hypoxia, usually associated with necrotic areas, in inflammation, and infections, where T lymphocyte capability of producing stimulatory cytokines is desirable.     

Impact of hypoxia and long-term cultivation on the genomic stability and mitochondrial performance of ex vivo expanded human stem/stromal cells

Recent studies have described the occurrence of chromosomal abnormalities and mitochondrial dysfunction in human stem/stromal cells (SCs), particularly after extensive passaging in vitro and/or expansion under low oxygen tensions. To deepen this knowledge we investigated the influence of hypoxia (2% O(2)) and prolonged passaging (>P10) of human bone marrow stromal cells (BMSCs) and adipose-derived stromal cells (ASCs) on the expression of genes involved in DNA repair and cell-cycle regulation pathways, as well as on the occurrence of microsatellite instability and changes in telomere length. Our results show that hypoxic conditions induce an immediate and concerted down-regulation of genes involved in DNA repair and damage response pathways (MLH1, RAD51, BRCA1, and Ku80), concomitantly with the occurrence of microsatellite instability while maintaining telomere length. We further searched for mutations occurring in the mitochondrial genome, and monitored changes in intracellular ATP content, membrane potential and mitochondrial DNA content. Hypoxia led to a simultaneous decrease in ATP content and in the number of mitochondrial genomes, whereas the opposite effect was observed after prolonged passaging. Moreover, we show that neither hypoxia nor prolonged passaging significantly affected the integrity of the mitochondrial genome. Ultimately, we present evidence on how hypoxia selectively impacts the cellular response of BMSCs and ASCs, thus pointing for the need to optimize oxygen tension according to the cell source.     

Modulation of the in vitro angiogenic potential of human mesenchymal stromal cells from different tissue sources

Mesenchymal stromal cells (MSCs) have been widely exploited for the treatment of several conditions due to their intrinsic regenerative and immunomodulatory properties. MSC have demonstrated to be particularly relevant for the treatment of ischemic diseases, where MSC-based therapies can stimulate angiogenesis and induce tissue regeneration. Regardless of the condition targeted, recent analyses of MSC-based clinical trials have demonstrated limited benefits indicating a need to improve the efficacy of this cell product. Preconditioning MSC ex vivo through microenvironment modulation was found to improve MSC survival rate and thus prolong their therapeutic effect. This workstudy aims at enhancing the in vitro angiogenic capacity of a potential MSC-based medicinal product by comparing different sources of MSC and culture conditions. MSC from three different sources (bone marrow [BM], adipose tissue [AT], and umbilical cord matrix [UCM]) were cultured with xenogeneic-/serum-free culture medium under static conditions and their angiogenic potential was studied. Results indicated a higher in vitro angiogenic capacity of UCM MSC, compared with cells derived from BM and AT. Physicochemical preconditioning of UCM MSC through a microcarrier-based culture platform and low oxygen concentration (2% O₂, compared with atmospheric air) increased the in vitro angiogenic potential of the cultured cells. Envisaging the clinical manufacturing of an allogeneic, off-the-shelf MSC-based product, preconditioned UCM MSC maintain the angiogenic gene expression profile upon cryopreservation and delivery processes in the conditions of our study. These results are expected to contribute to the development of MSC-based therapies in the context of angiogenesis.