Endothelial Cell-conditioned Medium Research Articles

Isoflurane preconditioning inhibits the effects of tissue-type plasminogen activator on brain endothelial cell in an in vitro model of ischemic stroke.

Tissue-type plasminogen activator (tPA) is the only treatment for ischemic stroke. However, tPA could induce the intracranial hemorrhage (ICH), which is the main cause of death in ischemic stroke patient after tPA treatment. At present, there is no treatment strategy to ameliorate tPA-induced brain injury after ischemia. Therefore, we investigated the effect of pre-treated isoflurane, which is a volatile anesthetic and has beneficial effects on neurological dysfunction, brain edema and infarct volume in ischemic stroke model. In this study, we used oxygen/glucose deprivation and reperfusion (OGD/R) condition to mimic an ischemic stroke in vitro. Matrix metalloproteinases (MMP) activity was measured in endothelial cell media. Also, neuronal cell culture was performed to investigate the effect of pretreated isoflurane on the neuronal cell survival after tPA-induced injury during OGD/R. Isoflurane pretreatment prevented tPA-induced MMP-2 and MMP-9 activity and suppressed tPA-triggered LRP/NF-κB/Cox-2 signaling after OGD/R. Neuronal cells, incubated with endothelial cell conditioned medium (EC-CM) after tPA + OGD/R, showed upregulation of pro-apoptotic molecules. However, neurons incubated with isoflurane-pretreated EC-CM showed increased anti-apoptotic molecules. Our findings suggest that isoflurane pretreatment could attenuate tPA-exaggerated brain ischemic injury, by reducing tPA-induced LRP/NF-κB/Cox-2 in endothelial cells, endothelial MMP-2 and MMP-9 activation, and subsequent pro-apoptotic molecule in neurons after OGD/R.

Endothelial cells regulate β-catenin activity in adrenocortical cells via secretion of basic fibroblast growth factor

Endothelial cell-derived products influence the synthesis of aldosterone and cortisol in human adrenocortical cells by modulating proteins such as steroidogenic acute-regulatory (StAR) protein, steroidogenic factor (SF)-1 and CITED2. However, the potential endothelial cell-derived factors that mediate this effect are still unknown. The current study was perfomed to look into the control of β-catenin activity by endothelial cell-derived factors and to identify a mechanism by which they affect β-catenin activity in adrenocortical NCIH295R cells.Using reporter gene assays and Western blotting, we found that endothelial cell-conditioned medium (ECCM) led to nuclear translocation of β-catenin and an increase in β-catenin-dependent transcription that could be blocked by U0126, an inhibitor of the mitogen-activated protein kinase pathway. Furthermore, we found that a receptor tyrosin kinase (RTK) was involved in ECCM-induced β-catenin-dependent transcription. Through selective inhibition of RTK using Su5402, it was shown that receptors responding to basic fibroblast growth factor (bFGF) mediate the action of ECCM.Adrenocortical cells treated with bFGF showed a significant greater level of bFGF mRNA. In addition, HUVECs secrete bFGF in a density-dependent manner. In conclusion, the data suggest that endothelial cells regulate β-catenin activity in adrenocortical cells also via secretion of basic fibroblast growth factor.

Blockade of Apoptosis Signal-Regulating Kinase 1 Attenuates Matrix Metalloproteinase 9 Activity in Brain Endothelial Cells and the Subsequent Apoptosis in Neurons after Ischemic Injury

Conditions of increased oxidative stress including cerebral ischemia can lead to blood–brain barrier dysfunction via matrix metalloproteinase (MMP). It is known that MMP-9 in particular is released from brain endothelial cells is involved in the neuronal cell death that occurs after cerebral ischemia. In the intracellular signaling network, apoptosis signal-regulating kinase 1 (ASK1) is the main activator of the oxidative stress that is part of the pathogenesis of cerebral ischemia. ASK1 also promotes apoptotic cell death and brain infarction after ischemia and is associated with vascular permeability and the formation of brain edema. However, the relationship between ASK1 and MMP-9 after cerebral ischemia remains unknown. Therefore, the aim of the present study was to determine whether blocking ASK1 would affect MMP-9 activity in the ischemic brain and cultured brain endothelial cells. Our results showed that ASK1 inhibition efficiently reduced MMP-9 activity in vivo and in vitro. In endothelial cell cultures, ASK1 inhibition upregulated phosphatidylinositol 3-kinase/Akt/nuclear factor erythroid 2 [NF-E2]-related factor 2/heme oxygenase-1 signals and downregulated cyclooxygenase-2 signals after hypoxia/reperfusion. Additionally, in neuronal cell cultures, cell death occurred when neurons were incubated with endothelial cell-conditioned medium (EC-CM) obtained from the hypoxia/reperfusion group. However, after incubation with EC-CM and following treatment with the ASK1 inhibitor NQDI-1, neuronal cell death was efficiently decreased. We conclude that suppressing ASK1 decreases MMP-9 activity in brain endothelial cells, and leads to decreased neuronal cell death after ischemic injury.

Endothelium-Derived 5-Methoxytryptophan Is a Circulating Anti-Inflammatory Molecule That Blocks Systemic Inflammation.

Systemic inflammation has emerged as a key pathophysiological process that induces multiorgan injury and causes serious human diseases. Endothelium is critical in maintaining cellular and inflammatory homeostasis, controlling systemic inflammation, and progression of inflammatory diseases. We postulated that endothelium produces and releases endogenous soluble factors to modulate inflammatory responses and protect against systemic inflammation. To identify endothelial cell-released soluble factors that protect against endothelial barrier dysfunction and systemic inflammation. We found that conditioned medium of endothelial cells inhibited cyclooxgenase-2 and interleukin-6 expression in macrophages stimulated with lipopolysaccharide. Analysis of conditioned medium extracts by liquid chromatography-mass spectrometry showed the presence of 5-methoxytryptophan (5-MTP), but not other related tryptophan metabolites. Furthermore, endothelial cell-derived 5-MTP suppressed lipopolysaccharide-induced inflammatory responses and signaling in macrophages and endotoxemic lung tissues. Lipopolysaccharide suppressed 5-MTP level in endothelial cell-conditioned medium and reduced serum 5-MTP level in the murine sepsis model. Intraperitoneal injection of 5-MTP restored serum 5-MTP accompanied by the inhibition of lipopolysaccharide-induced endothelial leakage and suppression of lipopolysaccharide- or cecal ligation and puncture-mediated proinflammatory mediators overexpression. 5-MTP administration rescued lungs from lipopolysaccharide-induced damages and prevented sepsis-related mortality. Importantly, compared with healthy subjects, serum 5-MTP level in septic patients was decreased by 65%, indicating an important clinical relevance. We conclude that 5-MTP belongs to a novel class of endothelium-derived protective molecules that defend against endothelial barrier dysfunction and excessive systemic inflammatory responses.

An endothelial cultured condition medium embedded porous PLGA scaffold for the enhancement of mouse embryonic stem cell differentiation

In this study, we have developed a microporous poly(lactic-co-glycolic acid) (PLGA) scaffold that combines a continuous release property and a three-dimensional (3D) scaffolding technique for the precise and efficient formation of endothelial cell lineage from embryonic stem cells (ESCs). Eight PLGA scaffolds (14.29%, 16.67%, 20% and 25% concentrations of PLGA solutions) mixed with two crystal sizes of sodium chloride (NaCl) were fabricated by leaching. Then, vascular endothelial cell conditioned medium (ECCM) mixed with gelatin was embedded into the scaffold for culturing of mouse embryonic stem cells (mESCs). The 14.29% PLGA scaffolds fabricated using non-ground NaCl particles (NG-PLGA) and the 25% PLGA containing scaffolds fabricated using ground NaCl particles (G-PLGA) possessed minimum and maximum moisture content and bovine serum albumin (BSA) content properties, respectively. These two groups of scaffolds were used for future experiments in this study. Cell culture results demonstrated that the proposed porous scaffolds without growth factors were sufficient to induce mouse ESCs to differentiate into endothelial-like cells in the early culture stages, and combined with embedded ECCM could provide a long-term inducing system for ESC differentiation.

Abstract A03: The effects of the cellular microenvironment on medulloblastoma metastasis

Abstract Medulloblastoma (MB) is the most common pediatric brain tumor, known to be highly malignant and invasive within the brain as well as extraneural locations. The mechanisms inducing MB metastasis and dissemination are not completely understood. Astrocytes, the most abundant cell type in the brain, have been shown in our lab to secrete factors that induce breast cancer brain metastasis (Wang et al., 2013). In addition to astrocytes, many brain cell types have also been implicated in the malignancy of brain tumors, including microglia, pericytes, and endothelial cells as part of the neurovascular unit. The overall goal of this study is to determine the effects of cells in the tumor microenvironment on MB metastasis. Our hypothesis is that astrocytes and/or other cell types in the neurovascular unit and microenvironment secrete factors that bring upon phenotypic changes in MB cells, leading to increased metastasis. To investigate this hypothesis, astrocyte conditioned media (ACM) from neonatal rat astrocyte cultures were used in in vitro assays to determine functional changes to Daoy MB cells. We observed that ACM (both serum free and serum depleted) in the lower chamber of a Boyden chamber Matrigel invasion assay induced invasion of Daoy cells more than 50-fold in comparison to DMEM in the lower well. Brain endothelial cell conditioned media also increased invasion of Daoy cells, which was comparable to ACM. ACM did not significantly affect proliferation or apoptosis of Daoy cells, as tested by BrdU incorporation and Caspase-3/-7 activity, respectively. When cultured appropriately in serum free ACM (+bFGF and EGF), Daoy cells were able to form larger and more proliferative neurospheres in comparison to DMEM, indicative of an increase in neural or cancer stem cell characteristics. To determine the effects of ACM on MB metastasis in vivo, we developed a zebrafish xenotransplant model. Daoy-GFP cells were injected into the hindbrain of 48 hour post fertilization Absolut FLK-mCherry zebrafish embryos. Using time-lapse confocal microscopy, we are able to image the cells and observe their movement within the microenvironment after injection. With this method we are working to determine changes in cell morphology and movement in Daoy-GFP cells that were cultured in ACM prior to injection. In order to further elucidate the mechanism in which ACM increases invasion and neurosphere formation, microarray studies are underway to pin-point a factor(s) or pathway(s) involved. Together, these data indicate that cells comprising the neurovascular unit, namely endothelial cells and astrocytes, contribute in a paracrine manner to MB metastasis and dissemination. Citation Format: Emily Gronseth, Ling Wang, Chris Koceja, Ramani Ramchandran. The effects of the cellular microenvironment on medulloblastoma metastasis. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A03.

Two Bioactive Molecular Weight Fractions of a Conditioned Medium Enhance RPE Cell Survival on Age-Related Macular Degeneration and Aged Bruch's Membrane.

PurposeTo characterize molecular weight fractions of bovine corneal endothelial cell conditioned medium (CM) supporting retinal pigment epithelium (RPE) cell survival on aged and age-related macular degeneration (AMD) Bruch's membrane.MethodsCM was subject to size separation using centrifugal filters. Retentate and filtrate fractions were tested for bioactivity by analyzing RPE survival on submacular Bruch's membrane of aged and AMD donor eyes and behavior on collagen I-coated tissue culture wells. Protein and peptide composition of active fractions was determined by mass spectrometry.ResultsTwo bioactive fractions, 3-kDa filtrate and a 10-50–kDa fraction, were necessary for RPE survival on aged and AMD Bruch's membrane. The 3-kDa filtrate, but not the 10-50–kDa fraction, supported RPE growth on collagen 1‐coated tissue culture plates. Mass spectrometry of the 10-50–kDa fraction identified 175 extracellular proteins, including growth factors and extracellular matrix molecules. Transforming growth factor (TGF)β-2 was identified as unique to active CM. Peptides representing 29 unique proteins were identified in the 3-KDa filtrate.ConclusionsThese results indicate there is a minimum of two bioactive molecules in CM, one found in the 3-kDa filtrate and one in the 10-50–kDa fraction, and that bioactive molecules in both fractions must be present to ensure RPE survival on Bruch's membrane. Mass spectrometry analysis suggested proteins to test in future studies to identify proteins that may contribute to CM bioactivity.Translational RelevanceResults of this study are the first steps in development of an adjunct to cell-based therapy to ensure cell transplant survival and functionality in AMD patients.

Retinoic acid-loaded polymeric nanoparticles enhance vascular regulation of neural stem cell survival and differentiation after ischaemia.

Stroke is one of the leading causes of death and disability worldwide. However, current therapies only reach a small percentage of patients and may cause serious side effects. We propose the therapeutic use of retinoic acid-loaded nanoparticles (RA-NP) to safely and efficiently repair the ischaemic brain by creating a favourable pro-angiogenic environment that enhances neurogenesis and neuronal restitution. Our data showed that RA-NP enhanced endothelial cell proliferation and tubule network formation and protected against ischaemia-induced death. To evaluate the effect of RA-NP on vascular regulation of neural stem cell (NSC) survival and differentiation, endothelial cell-conditioned media (EC-CM) were collected. EC-CM from healthy RA-NP-treated cells reduced NSC death and promoted proliferation while EC-CM from ischaemic RA-NP-treated cells decreased cell death, increased proliferation and neuronal differentiation. In parallel, human endothelial progenitor cells (hEPC), which are part of the endogenous repair response to vascular injury, were collected from ischaemic stroke patients. hEPC treated with RA-NP had significantly higher proliferation, which further highlights the therapeutic potential of this formulation. To conclude, RA-NP protected endothelial cells from ischaemic death and stimulated the release of pro-survival, proliferation-stimulating factors and differentiation cues for NSC. RA-NP were shown to be up to 83-fold more efficient than free RA and to enhance hEPC proliferation. These data serve as a stepping stone to use RA-NP as vasculotrophic and neurogenic agents for vascular disorders and neurodegenerative diseases with compromised vasculature.

MTR-05ANTI-ANGIOGENIC STRESS DRIVES A c-Met/ß1 INTEGRIN COMPLEX PROMOTING ALLOSTERIC ACTIVATION DRIVING INVASION

Invasive resistance limits anti-angiogenic therapy's efficacy. We previously reported upregulated c-Met and b1 integrin in bevacizumab-resistant glioblastomas. We hypothesized chemotactic c-Met/HGF and haptotactic b1 integrin physically interact, forming an alliance driving invasive growth. Immunoprecipitated lysates of intracranial xenografts of U87-BevR and U87-BevS, models of bevacizumab-resistance and responsiveness, revealed robust c-Met/b1 integrin interactions in bevacizumab-treated U87-BevRs, and minimal complex in bevacizumab-treated U87-BevS, IgG-treated U87-BevR, or U87-BevS. Proximity ligation assays (PLAs) mirrored results of our immunopreicipitation. In culture, complex formation increased dose-dependently by bevacizumab-induced VEGF depletion, exposure to c-Met ligand HGF, and hypoxia. Complex formation was inhibited by VEGF165, the predominant VEGF isoform, and endothelial cell conditioned medium. Far-western blotting demonstrated only extracellular c-Met domain to bind b1 integrin. The c-Met/b1 integrin interaction led to cross-activation, with increasing concentrations of b1 integrin ligand fibronectin which increased c-Met phosphorylation. This was inhibited with integrin-linked kinase (ILK) knockdown. Complex induction via iDimerize, where b1 integrin/c-Met were tagged with complementary proteins, forming a complex upon heterodimer addition, increased migration substantially (P < 0.05). Site-directed biopsy of bevacizumab-resistant glioblastoma patient revealed increased complex formation progressing from core to enhancing edge to invasive cells outside enhancement. PLAs revealed 20-fold more c-Met/b1 complexes after bevacizumab resistance in patient-specimens compared to bevacizumab-naive glioblastomas at recurrence (P < 0.05). Furthermore, the percentage of cMet/b1 integrin from 20 patients taken at diagnosis correlated inversely with survival (P < 0.05). Anti-angiogenic therapy dose on complex formation in vivo by treating xenografts with 3 mg/kg bevacizumab was as effective as 10 mg/kg; however, only the latter contained c-Met/b1 complexes. A patient receiving 5mg/kg of bevacizumab, had no increased complex under PLA. In summary, we have demonstrated a mechanism where b1 integrin forms a complex with c-Met in bevacizumab-resistant glioblastoma, leading to allosteric cross-activation, driving the invasive resistant phenotype.

Human placenta-derived multipotent mesenchymal stromal cells involved in placental angiogenesis via the PDGF-BB and STAT3 pathways.

We studied the smooth muscle cell differentiation capability of human placental multipotent mesenchymal stromal cells (hPMSCs) and identified how endothelial cells recruit hPMSCs participating in vessel formation. hPMSCs from term placentas were induced to differentiate into smooth muscle cells under induction conditions and different matrix substrates. We assessed endothelial cells from umbilical veins for platelet-derived growth factor (PDGF)-BB expression and to induce hPMSC PDGFR-beta and STAT3 activation. Endothelial cells were co-cultured with hPMSCs for in vitro angiogenesis. Cell differentiation ability was then further assessed by mouse placenta transplantation assay. hPMSCs can differentiate into smooth muscle cells; collagen type I and IV or laminin support this differentiation. Endothelial cells expressed significant levels of PDGF-BB and activated STAT3 transcriptional activity in hPMSCs. Endothelial cell-conditioned medium induced hPMSC migration, which was inhibited by STAT3 small interfering RNA transfection or by pretreatement with PDGFR-beta-blocking antibody but not by PDGFR-alpha-blocking antibody or isotype immunoglobulin G (IgG; P < 0.001). hPMSCs can incorporate into endothelial cells with tube formation and promote endothelial cells, forming capillary-like networks than endothelial cells alone (tube lengths: 12 024.1 ± 960.1 vs. 9404.2 ± 584.7 pixels; P < 0.001). Capillary-like networks were significantly reduced by hPMSCs pretreated with PDGFR-beta-blocking antibody but not by PDGFR-alpha-blocking antibody or isotype IgG (P < 0.001). Transplantation of hPMSCs into mouse placentas revealed incorporation of the hPMSCs into vessel walls, which expressed alpha-smooth muscle actin, calponin, and smooth muscle myosin (heavy chain) in vivo. In conclusion, endothelial cell-hPMSC interactions occur during vessel development of placenta. Placental endothelial cell-derived PDGF-BB recruits hPMSCs involved in vascular development via PDGFR-beta/STAT3 activation.

Human Embryonic Stem Cell–Derived Endothelial Precursor Cell Conditioned Medium Reduces the Thickness of the Capsule Around Silicone Implants in Rats

We postulated that the fibrotic capsule around a silicone implant could be induced by ischemic conditions, based on previous reports that hypoxia is an important microenvironmental factor in the development of tissue fibrosis. This study aimed to determine the effect of human embryonic stem cell (hESC)-derived endothelial precursor cell (EPC) conditioned medium (CM), which has strong angiogenic potential, on the development of capsule around the silicone implant in a rat model. Three groups had a mini-silicone implant with a smooth surface inserted. In 2 experimental groups, hESC-EPC CM was administered into the subcutaneous pocket either 1 or 2 times. After 2 months, the capsules were harvested and analyzed by histologic examination. There was a significant reduction in the thickness of the peri-implant capsules (P < 0.05) between the control and experimental groups. There is no tendency that hESC-EPC CM reduces inflammatory reaction in early postoperative periods. The experimental group showed increased angiogenesis compared to the control group (P < 0.05). Tissue hypoxia around the implant may be another cause for the peri-implant capsule. A preventive or therapeutic strategy to decrease capsular contracture by relieving the ischemic condition around the implant can be investigated in the future.

Muscle cell derived angiopoietin-1 contributes to both myogenesis and angiogenesis in the ischemic environment.

Recent strategies to treat peripheral arterial disease (PAD) have focused on stem cell based therapies, which are believed to result in local secretion of vascular growth factors. Little is known, however, about the role of ischemic endogenous cells in this context. We hypothesized that ischemic muscle cells (MC) are capable of secreting growth factors that act as potent effectors of the local cellular regenerative environment. Both muscle and endothelial cells (ECs) were subjected to experimental ischemia, and conditioned medium (CM) from each was collected and analyzed to assess myogenic and/or angiogenic potential. In muscle progenitors, mRNA expression of VEGF and its cognate receptors (Nrp1, Flt, Flk) was present and decreased during myotube formation in vitro, and EC CM or VEGF increased myoblast proliferation. Angiopoietin-1 (Ang-1), Tie1, and Tie2 mRNA increased during MC differentiation in vitro. Exogenous Ang-1 enhanced myogenic (MyoD and Myogenin) mRNA in differentiating myoblasts and increased myosin heavy chain protein. Myotube formation was enhanced by MC CM and inhibited by EC CM. Ang-1 protein was present in CM from MCs isolated from both the genetically ischemia-susceptible BALB/c and ischemia-resistant C57BL/6 mouse strains, and chimeric Tie2 receptor trapping in situ ablated Ang-1's myogenic effects in vitro. Ang-1 or MC CM enhanced myotube formation in a mixed isolate of muscle progenitors as well as a myoblast co-culture with pluripotent mesenchymal cells (10T1/2) and this effect was abrogated by viral expression of the extracellular domain of Tie2 (AdsTie2). Furthermore, mesh/tube formation by HUVECs was enhanced by Ang-1 or MC CM and abrogated by Tie2 chimeric receptor trapping. Our results demonstrate the ability of muscle and endothelial cell-derived vascular growth factors, particularly Ang-1, to serve as multi-functional stimuli regulating crosstalk between blood vessels and muscle cells during regeneration from ischemic myopathy.

Oxygen regulates human cytotrophoblast migration by controlling chemokine and receptor expression

IntroductionPlacental development involves the variation of oxygen supply due to vascular changes and cytotrophoblast invasion. Chemokines and their receptors play an important role during placental formation. Herein, the analysis of the chemokine/receptor pair CXCL12/CXCR4 and further chemokine receptors, such as CCR1, CCR7 and CXCR6 expression in human cytotrophoblasts was conducted. MethodsHuman cytotrophoblasts were examined directly after isolation or after incubation with different oxygen tensions and a chemical HIF-stimulator for 12 h with realtime PCR, immunoblot, immunohistochemistry. Conditioned media of placental villi, decidua, and endothelial cells was used for ELISA analysis of CXL12. Cytotrophoblast migration assays were conducted applying conditioned media of endothelial cells, a CXCL12 gradient, and different oxygen level. Endometrial and decidual tissue was stained for CXCL12 expression. ResultsAn upregulation of CXCL12, CXCR4, CCR1, CCR7 and CXCR6 was observed after cytotrophoblast differentiation. Low oxygen supply upregulated CXCR4, CCR7 and CXCR6, but downregulated CXCL12 and CCR1. In contrast to the HIF associated upregulation of the aforementioned proteins, downregulation of CXCL12 and CCR1 seemed to be HIF independent. Cytotrophoblast migration was stimulated by low oxygen, the application of a CXCL12 gradient and endothelial cell conditioned media. CXCL12 was detected in endometrial vessels, glands and conditioned media of placental and decidual tissue, but not decidual vessels. Discussion/conclusionTaken together, oxygen supply and cytotrophoblast differentiation seem to be regulators of chemokine and receptor expression and function in human cytotrophoblasts. Therefore, this system seems to be involved in placental development, directed cytotrophoblast migration in the decidual compartment and a subsequent sufficient supply of the growing fetus.

Endothelial cell-specific reactive oxygen species production increases susceptibility to aortic dissection.

Increased production of reactive oxygen species (ROS) throughout the vascular wall is a feature of cardiovascular disease states, but therapeutic strategies remain limited by our incomplete understanding of the role and contribution of specific vascular cell ROS to disease pathogenesis. To investigate the specific role of endothelial cell (EC) ROS in the development of structural vascular disease, we generated a mouse model of endothelium-specific Nox2 overexpression and tested the susceptibility to aortic dissection after angiotensin II (Ang II) infusion. A specific increase in endothelial ROS production in Nox2 transgenic mice was sufficient to cause Ang II-mediated aortic dissection, which was never observed in wild-type mice. Nox2 transgenic aortas had increased endothelial ROS production, endothelial vascular cell adhesion molecule-1 expression, matrix metalloproteinase activity, and CD45(+) inflammatory cell infiltration. Conditioned media from Nox2 transgenic ECs induced greater Erk1/2 phosphorylation in vascular smooth muscle cells compared with wild-type controls through secreted cyclophilin A (CypA). Nox2 transgenic ECs (but not vascular smooth muscle cells) and aortas had greater secretion of CypA both at baseline and in response to Ang II stimulation. Knockdown of CypA in ECs abolished the increase in vascular smooth muscle cell Erk1/2 phosphorylation conferred by EC conditioned media, and preincubation with CypA augmented Ang II-induced vascular smooth muscle cell ROS production. These findings demonstrate a pivotal role for EC-derived ROS in the determination of the susceptibility of the aortic wall to Ang II-mediated aortic dissection. ROS-dependent CypA secretion by ECs is an important signaling mechanism through which EC ROS regulate susceptibility of structural components of the aortic wall to aortic dissection.

Transdifferentiation of hypertrophic chondrocytes during endochondral bone repair by activation of pluripotent stem cell programs (216.1)

The majority of bone fractures heal by endochondral ossification, where bone forms secondarily to cartilage. Mechanistically bone repair is presumed to parallel endochondral ossification during development. Dogma holds that chondrocytes mature to hypertrophy, undergo apoptosis, and bone forms by invading osteoprogenitors. However, we have data to suggest chondrocytes transform directly into bone during regeneration. Consequently, we propose an alternate hypothesis where transdifferentiation of chondrocytes into osteocytes plays a direct role in endochondral bone regeneration. Using lineage tracing to follow cartilage transplanted into segmental bone defects created in murine tibiae, we demonstrate the vast majority of osteocytes are cartilage derived. Hypertrophic chondrocytes in the fracture callus are mineralized and express classic bone markers, without significant apoptosis. Instead some “transitioning” chondrocytes express Oct4A and sox‐2, suggesting that activation of the pluripotent programs may facilitate phenotypic transformation. Histologically this process appears coordinated by the invading vasculature and mineralization of cartilage can be fully recapitulated in vitro using endothelial cell conditioned medium. Taken together, these data suggest that there may be significant differences between endochondral ossification during bone development and repair.Grant Funding Source: NIH NRSA F32 (Bahney), MTF Junior Investigator Award (Bahney), NIH #AR053645 &amp; AR057 (TM)

Functional Analysis of Two Haplotypes of the Human Endothelial Protein C Receptor Gene

To confirm the effect of the endothelial protein receptor gene (PROCR) haplotypes H1 and H3 on venous thromboembolism (VTE), to study their effect on endothelial protein C receptor (EPCR) expression in human umbilical vein endothelial cells, and to investigate the functionality of H1 tagging single-nucleotide polymorphisms in an in vitro model. Protein C (PC), activated PC, and soluble EPCR (sEPCR) levels were measured in 702 patients with VTE and 518 healthy individuals. All subjects were genotyped for PROCR H1 and H3. Human umbilical vein endothelial cells isolated from 111 umbilical cords were used to study the relation between PROCR haplotypes, PROCR mRNA, cellular distribution of EPCR, and rate of PC activation. Finally, the functionality of the intragenic PROCR H1 single-nucleotide polymorphisms was analyzed using a luciferase-based method. We confirmed that individuals carrying H1 have reduced VTE risk, increased plasma activated PC levels, and reduced plasma sEPCR levels and that individuals with the H3H3 genotype have an increased VTE risk and increased plasma sEPCR levels. In cultured human umbilical vein endothelial cells, H1 is associated with increased membrane-bound EPCR, increased rate of PC activation, and reduced sEPCR in conditioned medium, but does not significantly influence PROCR mRNA levels. In contrast, H3 is associated with reduced membrane-bound EPCR and increased sEPCR in human umbilical vein endothelial cell-conditioned medium, higher levels of a truncated mRNA isoform, and a lower rate of PC activation. Finally, we identified the g.2132T>C single-nucleotide polymorphism in intron 1 as an intragenic H1-specific functional single-nucleotide polymorphism. These results support a protective role of PROCR H1 against VTE and an increased risk of VTE associated with the H3 haplotype.

Biochemical restoration of aged human Bruch's membrane: experimental studies to improve retinal pigment epithelium transplant survival and differentiation.

Suspensions of human embryonic stem cell-derived retinal pigment epithelium (hES-RPE) and human fetal RPE resurface aged and age-related macular degeneration (AMD) Bruch's membrane to a limited degree at day 21 in organ culture. Survival and differentiation of hES-RPE and human fetal RPE on aged or AMD Bruch's membrane are enhanced greatly (200%) if a biologically synthesized extracellular matrix (bovine corneal endothelial cell extracellular matrix) is laid down on Bruch's membrane prior to transplantation. Transplanted RPE survival is enhanced even more (400-1,000%) if Bruch's membrane is treated with bovine corneal endothelial cell-conditioned medium during organ culture of hES-RPE or fetal RPE on aged or AMD Bruch's membrane. Future efforts are focused on identifying the bioactive components of bovine corneal endothelial cell-conditioned medium, so that this material can be reconstituted for clinical use as an adjunct to improve RPE transplant survival and differentiation in AMD eyes.

Loss of menin mediated by endothelial cells treated with CoPP is associated with increased maturation of adipocytes

Oxidative stress is caused by an increase in reactive oxygen species (ROS) relative to the antioxidant defense system. An increase in ROS is known to decrease vascular function, increase inflammatory cytokines, and promote adipocyte hypertrophy. A known regulator of the oxidative stress response is the heat shock protein, heme-oxygenase 1 (HO-1), which is induced by cobalt protoporphyrin IX (CoPP). Menin was recently found to promote the sustained expression of heat shock proteins and is implicated in the regulation of oxidative stress. In this study, we investigated how changes in menin expression affected adipogenesis via the interaction between endothelial cells and adipocytes in response to CoPP treatment during oxidative stress. Using angiotensin II (Ang II) to induce oxidative stress in endothelial cells and adipocytes, we observed the induction of various cytokines including EGF, VEGF, angiogenin, IL-6, and MCP-1. Preadipocytes cultured in endothelial cell conditioned media treated with Ang II showed no changes in differentiation markers. Preadipocytes treated with the endothelial cell-conditioned media pretreated with CoPP resulted in an increase in the number of adipocytes, which expressed higher levels of adipocyte differentiation markers in direct correlation with the complete downregulation of the stress response regulator, menin. This change was not detected in adipocytes directly treated with CoPP alone. Therefore, we concluded that loss of menin is associated with the maturation of adipocytes induced by conditioned media from endothelial cells treated with CoPP.

Hyperglycemia-Induced Secretion of Endothelial Heparanase Stimulates a Vascular Endothelial Growth Factor Autocrine Network in Cardiomyocytes That Promotes Recruitment of Lipoprotein Lipase

During diabetes mellitus, coronary lipoprotein lipase increases to promote the predominant use of fatty acids. We have reported that high glucose stimulates active heparanase secretion from endothelial cells to cleave cardiomyocyte heparan sulfate and release bound lipoprotein lipase for transfer to the vascular lumen. In the current study, we examined whether heparanase also has a function to release cardiomyocyte vascular endothelial growth factor (VEGF), and whether this growth factor influences cardiomyocyte fatty acid delivery in an autocrine manner. Acute, reversible hyperglycemia was induced in rats, and a modified Langendorff heart perfusion was used to separate the coronary perfusate from the interstitial effluent. Coronary artery endothelial cells were exposed to high glucose to generate conditioned medium, and VEGF release from isolated cardiomyocytes was tested using endothelial cell conditioned medium or purified active and latent heparanase. Autocrine signaling of myocyte-derived VEGF on cardiac metabolism was studied. High glucose promoted latent and active heparanase secretion into endothelial cell conditioned medium, an effective stimulus for releasing cardiomyocyte VEGF. Intriguingly, latent heparanase was more efficient than active heparanase in releasing VEGF from a unique cell surface pool. VEGF augmented cardiomyocyte intracellular calcium and AMP-activated protein kinase phosphorylation and increased heparin-releasable lipoprotein lipase. Our data suggest that the heparanase-lipoprotein lipase-VEGF axis amplifies fatty acid delivery, a rapid and adaptive mechanism that is geared to overcome the loss of glucose consumption by the diabetic heart. If prolonged, the resultant lipotoxicity could lead to cardiovascular disease in humans.

Bone Morphogenetic Protein-2/-4 Upregulation Promoted by Endothelial Cells in Coculture Enhances Mouse Embryoid Body Differentiation

Endothelial cells (ECs) provide inductive signals for cell differentiation in vivo. However, it is unknown if these cells promote such differentiation in vitro and the signals involved. We investigated whether ECs are able to enhance the differentiation of the three germ layers and the underlying mechanisms. We established a coculture system of mouse embryoid bodies (EBs) and ECs. Then, we analyzed the expression of markers representative of the three germ layers, such as PDX-1, proinsulin, insulin1 (endoderm), nestin, neurofilament light (ectoderm), CD31, cardiotin, and cardiac troponin I (mesoderm) in EBs cultured alone (controls) or with ECs. A significant increase of these markers was observed in EBs cocultured with ECs compared to controls. The cocultured EBs also exhibited more robust vascular networks similar to those EBs treated with bone morphogenetic protein-2 or -4 (BMP-2 or -4). Therefore, the role of these peptides in the differentiation was investigated. We found a significant upregulation of BMP-2/-4 and BMP receptor 1A in EBs treated with EC conditioned medium (EC-CM) at early or middle stages of EB development. Recombinant human BMP-2 and BMP-4 exerted similar effects than EC-CM in the expression of BMPs or in the upregulation of the three germ layer specific markers. BMP-2/-4 antagonists, such as noggin and chordin-like-1, respectively inhibited the EC-CM inductive effects. These results demonstrate that ECs enhance the differentiation in vitro of cells that derived from the three germ layers and that BMP-2/-4 play a central role in this process.