Hypoxia-conditioned Medium Research Articles

Secretive derived from hypoxia preconditioned mesenchymal stem cells promote cartilage regeneration and mitigate joint inflammation via extracellular vesicles

Secretome derived from mesenchymal stem cells (MSCs) have profound effects on tissue regeneration, which could become the basis of future MSCs therapies. Hypoxia, as the physiologic environment of MSCs, has great potential to enhance MSCs paracrine therapeutic effect. In our study, the paracrine effects of secretome derived from MSCs preconditioned in normoxia and hypoxia was compared through both in vitro functional assays and an in vivo rat osteochondral defect model. Specifically, the paracrine effect of total EVs were compared to that of soluble factors to characterize the predominant active components in the hypoxic secretome. We demonstrated that hypoxia conditioned medium, as well as the corresponding EVs, at a relatively low dosage, were efficient in promoting the repair of critical-sized osteochondral defects and mitigated the joint inflammation in a rat osteochondral defect model, relative to their normoxia counterpart. In vitro functional test shows enhancement through chondrocyte proliferation, migration, and matrix deposition, while inhibit IL-1β-induced chondrocytes senescence, inflammation, matrix degradation, and pro-inflammatory macrophage activity. Multiple functional proteins, as well as a change in EVs' size profile, with enrichment of specific EV-miRNAs were detected with hypoxia preconditioning, implicating complex molecular pathways involved in hypoxia pre-conditioned MSCs secretome generated cartilage regeneration.

Hypoxia Treatment of Adipose Mesenchymal Stem Cells Promotes the Growth of Dermal Papilla Cells via HIF-1α and ERK1/2 Signaling Pathways.

Dermal papilla cells (DPCs) cultured in vitro induce hair follicle formation. Using a hypoxic microenvironment to culture adipose mesenchymal stem cells (ADSCs) can promote hair follicle growth. However, the exact molecular mechanisms underlying this process remain unclear. In this study, ADSCs and DPCs from Arbas Cashmere goats were used. A hypoxic microenvironment promoted the proliferation of ADSCs and increased the pluripotency of ADSCs. The growth factors vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) were upregulated in ADSCs in the hypoxia-conditioned medium (Hypo-cm). Hypo-cm also enhanced the ability of DPCs to induce hair follicle formation. Inhibitors of the ERK1/2 signaling pathway caused the expressions of growth factors that increased in hypoxic microenvironments to decrease; moreover, hypoxia-inducible factor-1α (HIF-1α) increased the expression levels of VEGF, bFGF, and PDGF and inhibited the expression of bone morphogenic protein 7 (BMP7). In conclusion, these findings improve the theoretical basis for the development of gene therapy drugs for the treatment of alopecia areata and hair thinning.

Treatment of critical bone defects using calcium phosphate cement and mesoporous bioactive glass providing spatiotemporal drug delivery

Calcium phosphate cements (CPC) are currently widely used bone replacement materials with excellent bioactivity, but have considerable disadvantages like slow degradation. For critical-sized defects, however, an improved degradation is essential to match the tissue regeneration, especially in younger patients who are still growing. We demonstrate that a combination of CPC with mesoporous bioactive glass (MBG) particles led to an enhanced degradation in vitro and in a critical alveolar cleft defect in rats. Additionally, to support new bone formation the MBG was functionalized with hypoxia conditioned medium (HCM) derived from rat bone marrow stromal cells. HCM-functionalized scaffolds showed an improved cell proliferation and the highest formation of new bone volume. This highly flexible material system together with the drug delivery capacity is adaptable to patient specific needs and has great potential for clinical translation.

IL-11 and soluble VCAM-1 are important components of Hypoxia Conditioned Media and crucial for Mesenchymal Stromal Cells attraction.

Bone marrow stromal cells (BMSC) are highly attractive for tissue engineering due to their ability to differentiate into different cell types, to expand extensively in vitro and to release paracrine soluble factors with a high regenerative potential. They were observed to migrate towards the sites of injury in response to chemotactic signals in vivo. During the last years hypoxia has become a proven method to control proliferation, differentiation and multipotency of BMSC. Conditioned medium from hypoxia-treated BMSC (Hypoxia-conditioned Medium; HCM) has been shown to have various favorable properties on tissue regeneration - such as on cell recruitment, wound healing, angiogenesis and revascularization. Due to this regenerative potential many studies attempt to further characterize HCM and its main functional components. In this study we used HCM generated from umbilical cord mesenchymal stem cells (UC-MSC) instead of BMSC, because GMP-verified methods were used to isolate and cultivate the cells and ensure their constant quality. UC-MSC have a high regenerative potential and are still immunologically naive and therefore highly unlikely to cause an immune reaction. In our article we took the first steps to closer investigate the role of umbilical cord MSC-derived HCM components, namely stromal cell-derived factor 1 (SDF-1α), interleukin 11 (IL-11) and soluble vascular cell adhesion molecule 1 (sVCAM-1). Our results show previously unknown roles of IL-11 and sVCAM-1 in the attraction of BMSC. The synergistic effect of the investigated protein mixture consisting of IL-11, sVCAM-1 and SDF-1α as well as those recombinant proteins alone revealed a significantly higher chemoattractive capacity towards human BMSC compared to normoxic control medium. Both, the protein mixtures and proteins alone as well as UC-HCM showed an angiogenic effect by promoting the formation of significantly longer tubule structures and higher amounts of junctions and tubules compared to normoxic control medium. By showing the prominent upregulation of IL-11, sVCAM-1 and SDF-1α under hypoxic conditions compared to normoxic control and revealing their crucial role in migration of human BMSC we took a further step forward in characterization of the chemoattractive components of HCM.

Characterization of Naturally Occurring Bioactive Factor Mixtures for Bone Regeneration.

In this study, the bone-regenerative potential of bioactive factors derived from adipose tissue, platelet-rich plasma (PRP) and conditioned medium from hypoxia-treated human telomerase immortalized bone-marrow-derived mesenchymal stem cells (hTERT-MSC) was investigated in vitro with the aim to develop cost-effective and efficient bone substitutes for optimized regeneration of bone defects. Adipose tissue was harvested from human donors undergoing reconstructive surgery, and adipose tissue extract (ATE) was prepared. Platelet lysates (PL) were produced by repeated freeze-thaw cycles of PRP, and hypoxia-conditioned medium (HCM) was obtained by culturing human telomerase immortalized bone-marrow-derived mesenchymal stromal cells for 5 days with 1% O2. Besides analysis by cytokine and angiogenesis arrays, ELISA was performed. Angiogenic potential was investigated in cocultures of bone-marrow-derived (BM)-MSC and human umbilical vein endothelial cells. Multiple angiogenic proteins and cytokines were detected in all growth factor mixtures. HCM and ATE contained high amounts of angiogenin and CCL2/MCP-1, whereas PL contained high amounts of IGFBP-1. Culturing cells with HCM and ATE significantly increased specific ALP activity of BM-MSC as well as tubule length and junctions of endothelial networks, indicating osteogenic and angiogenic stimulation. To achieve a synergism between chemoattractive potential and osteogenic and angiogenic differentiation capacity, a combination of different growth factors appears promising for potential clinical applications.

The Secretome of Hypoxia Conditioned hMSC Loaded in a Central Depot Induces Chemotaxis and Angiogenesis in a Biomimetic Mineralized Collagen Bone Replacement Material.

The development of biomaterials with intrinsic potential to stimulate endogenous tissue regeneration at the site of injury is a main demand on future implants in regenerative medicine. For critical-sized bone defects, an in situ tissue engineering concept is devised based on biomimetic mineralized collagen scaffolds. These scaffolds are functionalized with a central depot loaded with a signaling factor cocktail, obtained from secretome of hypoxia-conditioned human mesenchymal stem cells (MSC). Therefore, hypoxia-conditioned medium (HCM)-production is standardized and adapted to achieve high signaling factor-yields; a concentration protocol based on dialysis and freeze-drying is established to enable the integration of sufficient and defined amounts into the depot. In humid milieu-as after implantation-signaling factors are released by forming a chemotactic gradient, inducing a directed migration of human bone marrow stroma cells (hBMSC) into the scaffold. Angiogenic potential, determined by coculturing human umbilical vein endothelial cells (HUVEC) with osteogenically induced hBMSC shows prevascular structures, which sprout throughout the interconnected pores in a HCM-concentration-dependent manner. Retarded release by alginate-based (1 vol%) depots, significantly improves sprouting-depth and morphology of tubular structures. With the intrinsic potential to supply attracted cells with oxygen and nutrients, this bioactive material system has great potential for clinical translation.

Hypoxia enhances self-renewal properties and markers of mesenchymal stem cells

BackgroundMesenchymal stem cells (MSCs) are multipotent stromal cells that express CD73, CD90, and CD105 surface markers, but not CD14, CD45, CD34, CD11b, and HLA-DR. MSCs under hypoxic conditions have the essential role of maintaining the stemness capacity by releasing several growth factors into their medium, known as hypoxia conditioned medium (HCM). This study was performed to compare the effect of percentage of HCM to normoxic medium (NM) in increasing MSC proliferation marked by proliferation rate and surface marker expression.MethodsThis study was of post-test only control group design using human umbilical cord-MSCs (hUC-MSCs) as subjects. The HCM treatment group was obtained by culturing MSCs under 5% O2, whereas the NM control group was grown under 20% O2. The hUC-MSCs were divided into 4 groups with different dose ratios of HCM to NM (25%:75%; 50%:50%; 75%:25% for P1, P2 and P3, respectively and 100% of NM for the controls). All of these groups were maintained at 37oC and the data was collected after 72 hours incubation. MSC marker expression of CD73, CD90 and CD105 was analyzed using flow cytometry and MSC proliferation by trypan blue assay. ResultThere were significant differences in MSC marker expression of CD73, CD90 and CD105 and proliferation at all dose ratios of HCM to NM (p<0.05).ConclusionLow oxygen concentration promotes MSC proliferation and stemness thus it might be beneficial for maintaining the MSC physiologic niche in-vitro.

Anti-neovascularization effects of DMBT in age-related macular degeneration by inhibition of VEGF secretion through ROS-dependent signaling pathway.

Choroidal neovascularization (CNV) is the hallmark of late-staged wet age-related macular degeneration (AMD). Vascular endothelial growth factor (VEGF) is a key component in the development and progression of wet AMD. DMBT, 6,6'-bis(2,3-dimethoxybenzoyl)-α,α-D-trehalose, had been proved that it could suppress tumor angiogenesis and metastasis by inhibiting production of VEGF. But the effects of DMBT on CNV were not known. This study was to investigate effects and mechanisms of DMBT on CNV in vitro and in vivo. Results showed that DMBT could inhibit migration and tube formation of RF/6A cells under ARPE-19 hypoxia conditioned medium. DMBT could reduce lesion area in laser-induced CNV model mice. ELISA and Western blotting assay showed that DMBT markedly inhibited secretion of VEGF in vitro and in vivo. Furthermore, DMBT restrained ROS level under hypoxia via suppressing Nrf2/HO-1 pathway. DMBT effectively suppressed hypoxia-induced the up-regulation of p-Akt, p-NF-κB, and HIF-1α. These results suggest that DMBT can inhibit CNV by down-regulation of VEGF in retina through Akt/NF-κB/HIF-1α and ERK/Nrf2/HO-1/HIF-1α pathway. DMBT might be a promising lead molecule for anti-CNV and serve as a therapeutic agent to inhibit CNV.

Adipose Mesenchymal Stem Cell Secretome Modulated in Hypoxia for Remodeling of Radiation-Induced Salivary Gland Damage

Background and PurposeThis study was conducted to determine whether a secretome from mesenchymal stem cells (MSC) modulated by hypoxic conditions to contain therapeutic factors contributes to salivary gland (SG) tissue remodeling and has the potential to improve irradiation (IR)-induced salivary hypofunction in a mouse model.Materials and MethodsHuman adipose mesenchymal stem cells (hAdMSC) were isolated, expanded, and exposed to hypoxic conditions (O2 < 5%). The hypoxia-conditioned medium was then filtered to a high molecular weight fraction and prepared as a hAdMSC secretome. The hAdMSC secretome was subsequently infused into the tail vein of C3H mice immediately after local IR once a day for seven consecutive days. The control group received equal volume (500 μL) of vehicle (PBS) only. SG function and structural tissue remodeling by the hAdMSC secretome were investigated. Human parotid epithelial cells (HPEC) were obtained, expanded in vitro, and then irradiated and treated with either the hypoxia-conditioned medium or a normoxic control medium. Cell proliferation and IR-induced cell death were examined to determine the mechanism by which the hAdMSC secretome exerted its effects.ResultsThe conditioned hAdMSC secretome contained high levels of GM-CSF, VEGF, IL-6, and IGF-1. Repeated systemic infusion with the hAdMSC secretome resulted in improved salivation capacity and increased levels of salivary proteins, including amylase and EGF, relative to the PBS group. The microscopic structural integrity of SG was maintained and salivary epithelial (AQP-5), endothelial (CD31), myoepithelial (α-SMA) and SG progenitor cells (c-Kit) were successfully protected from radiation damage and remodeled. The hAdMSC secretome strongly induced proliferation of HPEC and led to a significant decrease in cell death in vivo and in vitro. Moreover, the anti-apoptotic effects of the hAdMSC secretome were found to be promoted after hypoxia-preconditioning relative to normoxia-cultured hAdMSC secretome.ConclusionThese results show that the hAdMSC secretome from hypoxic-conditioned medium may provide radioprotection and tissue remodeling via release of paracrine mediators.

Hypoxia Exerts Dualistic Effects on Inflammatory and Proliferative Responses of Healthy and Asthmatic Primary Human Bronchial Smooth Muscle Cells

BackgroundFor oxygen supply, airway wall cells depend on diffusion though the basement membrane, as well as on delivery by micro-vessels. In the asthmatic lung, local hypoxic conditions may occur due to increased thickness and altered composition of the basement membrane, as well as due to edema of the inflamed airway wall.ObjectiveIn our study we investigated the effect of hypoxia on proliferation and pro-inflammatory and pro-angiogenic parameter production by human bronchial smooth muscle cells (BSMC). Furthermore, conditioned media of hypoxia-exposed BSMC was tested for its ability to induce sprout outgrowth from endothelial cells spheroids.MethodsBSMC were cultured in RPMI1640 (5% FCS) under normoxic (21% O2) and hypoxic (1% and 5% O2) conditions. Proliferation was determined by cell count and Western blot analysis for cyclin E and Proliferating Cell Nuclear Antigen (PCNA). Secretion of IL-6, IL-8, ENA-78 and VEGF-A was analyzed by ELISA. BSMC conditioned medium was tested for its angiogenic capacity by endothelial cell (EC)-spheroid in vitro angiogenesis assay.ResultsProliferation of BSMC obtained from asthmatic and non-asthmatic patients was significantly reduced in the presence of 1% O2, whereas 5% O2 reduced proliferation of asthmatic BSMC only. Hypoxia induced HIF-1α expression in asthmatic and non-asthmatic BSMC, which coincided with significantly increased release of IL-6, IL-8 and VEGF-A, but not ENA-78. Finally, endothelial sprout outgrowth from EC spheroids was increased when exposed to hypoxia conditioned BSMC medium.ConclusionHypoxia had dualistic effects on proliferative and inflammatory responses of asthmatic and non-asthmatic BSMC. First, hypoxia reduced BSMC proliferation. Second, hypoxia induced a pro-inflammatory, pro-angiogenic response. BSMC and EC may thus be promising new targets to counteract and/or alleviate airway wall remodeling.

Hypoxia-inducible Factor-2α-dependent Hypoxic Induction of Wnt10b Expression in Adipogenic Cells

Adipocyte hyperplasia and hypertrophy in obesity can lead to many changes in adipose tissue, such as hypoxia, metabolic dysregulation, and enhanced secretion of cytokines. In this study, hypoxia increased the expression of Wnt10b in both human and mouse adipogenic cells, but not in hypoxia-inducible factor (HIF)-2α-deficient adipogenic cells. Chromatin immunoprecipitation analysis revealed that HIF-2α, but not HIF-1α, bound to the Wnt10b enhancer region as well as upstream of the Wnt1 gene, which is encoded by an antisense strand of the Wnt10b gene. Hypoxia-conditioned medium (H-CM) induced phosphorylation of lipoprotein-receptor-related protein 6 as well as β-catenin-dependent gene expression in normoxic cells, which suggests that H-CM contains canonical Wnt signals. Furthermore, adipogenesis of both human mesenchymal stem cells and mouse preadipocytes was inhibited by H-CM even under normoxic conditions. These results suggest that O2 concentration gradients influence the formation of Wnt ligand gradients, which are involved in the regulation of pluripotency, cell proliferation, and cell differentiation.

MUC1 enhances hypoxia-driven angiogenesis through the regulation of multiple proangiogenic factors

Pancreatic cancer is one of the most lethal malignancies due to its aggressive growth and rapid development of distant metastases. In this context, mucin 1 (MUC1) overexpression and hypoxia are frequently observed events. However, their functional relationship remains largely unknown. This study provides evidence that MUC1 is overexpressed by hypoxia and contributes to hypoxia-driven angiogenesis. Using the conditioned medium obtained from hypoxia-stressed AsPC1 cells treated with MUC1 siRNAs, we demonstrated that MUC1 enhanced the endothelial tube formation, proliferation and migration ability, which induced by hypoxia-conditioned medium (HCM). In addition, MUC1 was significantly induced by hypoxia, especially in the pancreatic cancer cells derived from metastatic tumors (AsPC1, HPAF2 or Capan1), and MUC1-cytoplasmic tail (MUC1-CT) accumulated in the nucleus under hypoxia. As noted in a previous report, MUC1-CT was recruited to genomic regions upstream of the connective tissue growth factor (CTGF) accompanied with β-catenin and p53, resulting in the hypoxic induction of CTGF. Moreover, hypoxia-induced MUC1 partially regulated two other hypoxia-inducible proangiogenic factors including vascular endothelial growth factor-A and platelet-derived growth factor-B. The neutralization assay revealed that endothelial tube formation induced by HCM was clearly suppressed by antibodies against these three factors, suggesting the importance of these factors in hypoxia-driven angiogenesis. In summary, this is the first report demonstrating a pivotal role of MUC1 in controlling the hypoxia-driven angiogenesis through the regulation of multiple proangiogenic factors in pancreatic cancer. Our findings provide the novel insights into the understanding of complex interactions between pancreatic cancer cells and tumor microenvironments.

Soluble Factors from Multipotent Mesenchymal Stromal Cells have Antinecrotic Effect on Cardiomyocytes in Vitro and Improve Cardiac Function in Infarcted Rat Hearts

The mechanisms underlying the functional improvement after injection of multipotent mesenchymal stromal cells (MSCs) in infarcted hearts remain incompletely understood. The aim of this study was to investigate if soluble factors secreted by MSCs promote cardioprotection. For this purpose, conditioned medium (CM) was obtained after three passages from MSC cultures submitted to 72 h of conditioning in serum-free DMEM under normoxia (NCM) or hypoxia (HCM) conditions. CM was concentrated 25-fold before use (NCM-25X, concentrated normoxia conditioned medium; HCM-25X, concentrated hypoxia conditioned medium). The in vitro cardioprotection was evaluated in neonatal ventricular cardiomyocytes by quantifying apoptosis after 24 h of serum deprivation associated with hypoxia (1% O(2)) in the absence or presence of NCM and HCM (nonconcentrated and 25-fold concentrated). The in vivo cardioprotection of HCM was tested in a model of myocardial infarction (MI) induced in Wistar male rats by permanent left coronary occlusion. Intramyocardial injection of HCM-25X (n = 14) or nonconditioned DMEM (n = 16) was performed 3 h after coronary occlusion and cardiac function was evaluated 19-21 days after medium injection. Cardiac function was evaluated by electro- and echocardiogram, left ventricular catheterization, and treadmill test. The in vitro results showed that HCM was able to decrease cardiomyocyte necrosis. The in vivo results showed that HCM-25X administered 3 h after AMI was able to promote a significant reduction (35%) in left ventricular end-diastolic pressure and improvement of cardiac contractility (15%) and relaxation (12%). These results suggest that soluble factors released in vitro by MSCs are able to promote cardioprotection in vitro and improve cardiac function in vivo.

Mesenchymal stromal cell-conditioned medium prevents radiation-induced small intestine injury in mice

Effective therapy for radiation-induced intestinal injury is currently unavailable. Mesenchymal stromal cells (MSC) are expected to be useful in repairing intestinal damage caused by irradiation. We determined whether the MSC-derived bioactive components could protect radiation-induced small intestine injury in mice. Human umbilical cord (UC)-derived MSC were isolated, expanded and exposed to hypoxic conditions in vitro. The hypoxia-conditioned medium was ultrafiltrated with a 3-kDa molecular weight cut-off to prepare the high molecular weight fraction (HMWF). The effect of HMWF on the viability of irradiated rat intestinal epithelial cells (IEC-6) was examined by MTT(methyl thiazolyl tetrazolium) assay. HMWF was also delivered to BALB/C male mice by tail intravenous injection immediately after receiving local abdominal irradiation at a selected dose of 10 Gy. Animal body weight, survival and diarrhea were monitored for 30 days. The improvement of mice intestine structure, including epithelium thickness and villus height, was examined by histology. HMWF enhanced the viability of irradiated IEC-6 cells in vitro. Repeated infusion of HMWF for 7 days immediately after abdominal irradiation of 10 Gy ((60)Coγ-ray) increased the survival rate, decreased diarrhea occurrence and improved the small intestinal structural integrity of irradiated mice. MSC-derived bioactive components could be a novel therapeutic approach for the treatment of radiation-induced injury.

Reactive oxygen species released from hypoxic hepatocytes regulates MMP-2 expression in hepatic stellate cells.

Hypoxia is a common environmental stress factor and is associated with fibrogenesis. Matrix metalloproteinase-2 (MMP-2), produced by hepatic stellate cells (HSCs), plays an important role in liver fibrogenesis. However, inconsistent results have been reported on the impact of hypoxia on MMP-2 expression and activity in HSCs. We speculated that cell–cell interaction is involved in the regulation of MMP-2 expression and activity at low oxygen level in vivo. Therefore, in this report we investigated the mechanism by which hypoxic hepatocytes regulates MMP-2 expression in HSCs. Our results showed that the conditioned medium from hypoxia-treated rat hepatocytes strongly induced the expression of MMP-2 mRNA and protein in rat HSC-T6 cells. Reduced glutathione neutralized ROS released from hypoxic hepatocytes, leading to reduced MMP-2 expression in HSC-T6 cells. In addition, phospho-IκB-α protein level was increased in HSC-T6 cells treated with hypoxia conditioned medium, and NF-κB signaling inhibitor inhibited MMP-2 expression in HSC-T6 cells. Taken together, our data suggest that ROS is an important factor released by hypoxic hepatocytes to regulate MMP-2 expression in HSCs, and NF-κB signaling is crucially involved in ROS-induced MMP-2 expression in HSCs. Our findings suggest that strategies aimed at antagonizing the generation of ROS in hypoxic hepatocytes and inhibiting NF-κB signaling in HSCs may represent novel therapeutic options for liver fibrosis.

Hypoxia Enhances Sphingosine Kinase 2 Activity and Provokes Sphingosine-1-Phosphate-Mediated Chemoresistance in A549 Lung Cancer Cells

Hypoxia and signaling via hypoxia-inducible factor-1 (HIF-1) is a key feature of solid tumors and is related to tumor progression as well as treatment failure. Although it is generally accepted that HIF-1 provokes tumor cell survival and induces chemoresistance under hypoxia, HIF-1-independent mechanisms operate as well. We present evidence that conditioned medium obtained from A549 cells, incubated for 24 h under hypoxia, protected naive A549 cells from etoposide-induced cell death. Lipid extracts generated from hypoxia-conditioned medium still rescued cells from apoptosis induced by etoposide. Specifically, the bioactive lipid sphingosine-1-phosphate (S1P) not only was essential for cell viability of A549 cells but also protected cells from apoptosis. We noticed an increase in sphingosine kinase 2 (SphK2) protein level and enzymatic activity under hypoxia, which correlated with the release of S1P into the medium. Knockdown of SphK2 using specific small interfering RNA relieved chemoresistance of A549 cells under hypoxia and conditioned medium obtained from SphK2 knockdown cells was only partially protective. Coincubations of conditioned medium with VPC23019, a S1P(1)/S1P(3) antagonist, reduced protection of conditioned medium, with the further notion that p42/44 mitogen-activated protein kinase transmits autocrine or paracrine survival signaling downstream of S1P(1)/S1P(3) receptors. Our data suggest that hypoxia activates SphK2 to promote the synthesis and release of S1P, which in turn binds to S1P(1)/S1P(3) receptors, thus activating p42/44 mitogen-activated protein kinase to convey autocrine or paracrine protection of A549 cells.

Stem cell factor stimulates neurogenesis in vitro and in vivo

Cerebral ischemia stimulates neurogenesis in proliferative zones of the rodent forebrain. To identify the signaling factors involved, cerebral cortical cultures prepared from embryonic mouse brains were deprived of oxygen. Hypoxia increased bromodeoxyuridine (BrdU) incorporation into cells that expressed proliferation markers and immature neuronal markers and that lacked evidence of DNA damage or caspase-3 activation. Hypoxia-conditioned medium and stem cell factor (SCF), which was present in hypoxia-conditioned medium at increased levels, also stimulated BrdU incorporation into normoxic cultures. The SCF receptor, c-kit, was expressed in neuronal cultures and in neuroproliferative zones of the adult rat brain, and in vivo administration of SCF increased BrdU labeling of immature neurons in these regions. Cerebral hypoxia and ischemia may stimulate neurogenesis through trophic factors, including SCF.

Stem cell factor stimulates neurogenesis in vitro and in vivo

Cerebral ischemia stimulates neurogenesis in proliferative zones of the rodent forebrain. To identify the signaling factors involved, cerebral cortical cultures prepared from embryonic mouse brains were deprived of oxygen. Hypoxia increased bromodeoxyuridine (BrdU) incorporation into cells that expressed proliferation markers and immature neuronal markers and that lacked evidence of DNA damage or caspase-3 activation. Hypoxia-conditioned medium and stem cell factor (SCF), which was present in hypoxia-conditioned medium at increased levels, also stimulated BrdU incorporation into normoxic cultures. The SCF receptor, c-kit, was expressed in neuronal cultures and in neuroproliferative zones of the adult rat brain, and in vivo administration of SCF increased BrdU labeling of immature neurons in these regions. Cerebral hypoxia and ischemia may stimulate neurogenesis through trophic factors, including SCF.

Stem cell factor stimulates neurogenesis in vitro and in vivo.

Cerebral ischemia stimulates neurogenesis in proliferative zones of the rodent forebrain. To identify the signaling factors involved, cerebral cortical cultures prepared from embryonic mouse brains were deprived of oxygen. Hypoxia increased bromodeoxyuridine (BrdU) incorporation into cells that expressed proliferation markers and immature neuronal markers and that lacked evidence of DNA damage or caspase-3 activation. Hypoxia-conditioned medium and stem cell factor (SCF), which was present in hypoxia-conditioned medium at increased levels, also stimulated BrdU incorporation into normoxic cultures. The SCF receptor, c-kit, was expressed in neuronal cultures and in neuroproliferative zones of the adult rat brain, and in vivo administration of SCF increased BrdU labeling of immature neurons in these regions. Cerebral hypoxia and ischemia may stimulate neurogenesis through trophic factors, including SCF.

Tie2 Receptor Expression Is Stimulated by Hypoxia and Proinflammatory Cytokines in Human Endothelial Cells

The tyrosine kinase receptor Tie2 (also known as Tek) plays an important role in the development of the embryonic vasculature and persists in adult endothelial cells (ECs). Tie2 was shown to be upregulated in tumors and skin wounds, and its ligands angiopoietin-1 and -2, although they are not directly mitogenic, modulate neovascularization. To gain further insight into the regulation of Tie2, we have studied the effect of hypoxia and inflammatory cytokines, two conditions frequently associated with neoangiogenic processes, on Tie2 expression in human ECs. Exposure to 1% O(2) led to a time-dependent significant rise of Tie2 protein levels in human coronary microvascular endothelial cells (HCMECs) and dermal microvascular ECs (HMEC-1) (3.2- and 2.5-fold within 24 hours), which was reversible after reoxygenation, and induced a less marked increase in human umbilical vein ECs (HUVECs; 1.7-fold). Hypoxia-conditioned medium and D-deoxyglucose did not change Tie2 expression, but desferrioxamine and cobalt, which are known to mimic hypoxia-sensing mechanisms, induced Tie2 at ambient oxygen tensions. Tumor necrosis factor-alpha induced Tie2 in a time- and dose-dependent fashion in all 3 EC types (HUVEC, 2.3-fold; HMEC-1, 2. 8-fold; and HCMEC, 3.0-fold; 10 ng/mL, 24 hours). Enhanced expression was also found after exposure to interleukin-1beta (1 ng/mL). Changes in Tie2 protein levels were paralleled by changes in mRNA expression. In accordance with these in vitro findings, immunohistochemistry revealed focal upregulation of Tie2 in capillaries at the border of infarcted human and rat myocardium. In conclusion, the data show that hypoxia and inflammatory cytokines upregulate Tie2, which may contribute to the angiogenic response in ischemic tissues.