ABCC4 suppresses glioblastoma progression and recurrence by restraining cGMP-PKG signalling.

BACKGROUND: Cyclic nucleotides are critical mediators of cellular signalling in glioblastoma. However, the clinical relevance and mechanisms of regulating cyclic nucleotides in glioblastoma progression and recurrence have yet to be thoroughly explored. METHODS: In silico, mRNA, and protein level analyses identified the primary regulator of cyclic nucleotides in recurrent human glioblastoma. Lentiviral and pharmacological manipulations examined the functional impact of cyclic nucleotide signalling in human glioma cell lines and primary glioblastoma cells. An orthotopic xenograft mice model coupled with aspirin hydrogels verified the in vivo outcome of targeting cyclic nucleotide signalling. RESULTS: Elevated intracellular levels of cGMP, instead of cAMP, due to a lower substrate efflux from ATP-binding cassette sub-family C member 4 (ABCC4) is engaged in the recurrence of glioblastoma. ABCC4 gene expression is negatively associated with recurrence and overall survival outcomes in glioblastoma specimens. ABCC4 loss-of-function activates cGMP-PKG signalling, promoting malignancy in glioblastoma cells and xenografts. Hydrogels loaded with aspirin, inhibiting glioblastoma progression partly by upregulating ABCC4 expressions, augment the efficacy of standard-of-care therapies in orthotopic glioblastoma xenografts. CONCLUSION: ABCC4, repressing the cGMP-PKG signalling pathway, is a tumour suppressor in glioblastoma progression and recurrence. Aspirin hydrogels impede glioblastoma progression through ABCC4 restoration and constitute a viable translational approach.

Cellular and exosomal GPx1 are essential for controlling hydrogen peroxide balance and alleviating oxidative stress in hypoxic glioblastoma.

Tumor hypoxia promotes malignant progression and therapeutic resistance in glioblastoma partly by increasing the production of hydrogen peroxide (H2O2), a type of reactive oxygen species critical for cell metabolic responses due to its additional role as a second messenger. However, the catabolic pathways that prevent H2O2 overload and subsequent tumor cell damage in hypoxic glioblastoma remain unclear. Herein, we present a hypoxia-coordinated H2O2 regulatory mechanism whereby excess H2O2 in glioblastoma induced by hypoxia is diminished by glutathione peroxidase 1 (GPx1), an antioxidant enzyme detoxifying H2O2, via the binding of hypoxia-inducible factor-1α (HIF-1α) to GPx1 promoter. Depletion of GPx1 results in H2O2 overload and apoptosis in glioblastoma cells, as well as growth inhibition in glioblastoma xenografts. Moreover, tumor hypoxia increases exosomal GPx1 expression, which assists glioblastoma and endothelial cells in countering H2O2 or radiation-induced apoptosis in vitro and in vivo. Clinical data explorations further demonstrate that GPx1 expression was positively correlated with tumor grade and expression of HIF-1α, HIF-1α target genes, and exosomal marker genes; by contrast, it was inversely correlated with the overall survival outcome in human glioblastoma specimens. Our analyses validate that the redox balance of H2O2 within hypoxic glioblastoma is clinically relevant and could be maintained by HIF-1α-promoted or exosome-related GPx1.

Hypoxia-induced CXC chemokine ligand 14 expression drives protumorigenic effects through activation of insulin-like growth factor-1 receptor signaling in glioblastoma.

Hypoxic tumor microenvironment (HTM) promotes a more aggressive and malignant state in glioblastoma. However, little is known about the role and mechanism of CXC chemokine ligand 14 (CXCL14) in HTM-mediated glioblastoma progression. In this study, we report that CXCL14 expression correlated with poor outcomes, tumor grade, and hypoxia-inducible factor (HIF) expression in patients with glioblastoma. CXCL14 was upregulated in tumor cells within the hypoxic areas of glioblastoma. Hypoxia induced HIF-dependent expression of CXCL14, which promoted glioblastoma tumorigenicity and invasiveness in vitro and in vivo. Moreover, CXCL14 gain-of-function in glioblastoma cells activated insulin-like growth factor-1 receptor (IGF-1R) signal transduction to regulate the growth, invasiveness, and neurosphere formation of glioblastoma. Finally, systemic delivery of CXCL14 siRNA nanoparticles (NPs) with polysorbate 80 coating significantly suppressed tumor growth in vivo and extended the survival time in patient-derived glioblastoma xenografts. Together, these findings suggest that HIF-dependent CXCL14 expression contributes to HTM-promoted glioblastoma tumorigenicity and invasiveness through activation of the IGF-1R signaling pathway. CXCL14 siRNA NPs as an oligonucleotide drug can inhibit glioblastoma progression and constitute a translational path for the clinical treatment of glioblastoma patients.

Haloperidol Instigates Endometrial Carcinogenesis and Cancer Progression by the NF-κB/CSF-1 Signaling Cascade.

Haloperidol is a routine drug for schizophrenia and palliative care of cancer; it also has antitumor effects in several types of cancer. However, the role of haloperidol in endometrial cancer (EC) development is still unclear. Here, we show that chronic haloperidol treatment in clinically relevant doses induced endometrial hyperplasia in normal mice and promoted tumor growth and malignancy in mice with orthotopic EC. The pharmacokinetic study indicated that haloperidol highly accumulated in the uterus of mice. In vitro studies revealed that haloperidol stimulated the cellular transformation of human endometrial epithelial cells (HECCs) and promoted the proliferation, migration, and invasion of human endometrial carcinoma cells (HECCs) by activating nuclear factor kappa B (NF-κB) and its downstream signaling target, colony-stimulating factor 1 (CSF-1). Gain of function of CSF-1 promotes the cellular transformation of HEECs and the malignant progression of HECCs. Moreover, blockade of CSF-1 inhibited haloperidol-promoted EC progression in vitro and in vivo. A population-based cohort study of EC patients further demonstrated that the use of haloperidol was associated with increased EC-specific mortality. Collectively, these findings indicate that clinical use of haloperidol could potentially be harmful to female patients with EC.

Superhelical DNA liquid crystals from dendrimer-induced DNA compaction.

Electrostatic compaction of double stranded DNA induced by a positively charged poly(amidoamine) (PAMAM) dendrimer of generation four (G4) was found to produce two unique types of DNA mesophases, in which the DNA bent into superhelices packed in a tetragonal or hexagonal lattice. The structure formed at a lower dendrimer charge density was three-dimensionally (3D) ordered, as characterized by the P41212 space group with a 41 screw axis in a tetragonal arrangement, showing that the weakly bent DNA superhelices with a pitch length of ca. 5.0 nm possessed both identical handedness and phase conservation. The 3D ordered structure transformed into a 2D mesophase at a higher dendrimer charge density, wherein the strongly bent superhelices with a pitch length of ca. 4.0 nm organized in a hexagonal lattice without lateral coherence of helical trajectory. The counterion valency of the protonic acid that is used to charge the dendrimer was found to influence the phase diagram. Under a given dendrimer charge density, the complex with a multivalent acid-protonated dendrimer tended to form structures with less curved DNA, attesting that the driving force of charge matching was reduced by increasing the counterion valency of the dendrimer.

Gain of CXCR7 function with mesenchymal stem cell therapy ameliorates experimental arthritis via enhancing tissue regeneration and immunomodulation.

BACKGROUND: The major barriers to mesenchymal stem cell (MSC) therapy in rheumatoid arthritis (RA) are a low extent of tissue regeneration and insufficient immunomodulation after cell transplantation. In addition, the role of C-X-C chemokine receptor type 7 (CXCR7) and its mechanism of action in MSC-mediated osteogenic or chondrogenic differentiation and immunomodulation are unclear. METHODS: Gain of CXCR7 function on human MSCs was carried out by lentiviral vector-mediated CXCR7 overexpression or CXCR7 agonist, TC14012. These cells were determined the role and potential mechanisms for CXCR7-regulated MSC differentiation and immunomodulation using cellular and molecular assays. The therapeutic benefits in RA were investigated in rats with collagen-induced arthritis (CIA). RESULTS: CXCR7 was upregulated in MSCs during the induction of osteogenic or chondrogenic differentiation. Blockage of CXCR7 function inhibited osteogenic or chondrogenic differentiation of MSCs whereas gain of CXCR7 function had the opposite effects. Besides, MSCs with CXCR7 gain-of-function facilitated macrophage apoptosis and regulatory T cell differentiation in a co-culture system. Gain of CXCR7 function also promoted the production of anti-inflammatory soluble factors. A gene expression profiling assay and signaling reporter assays revealed that CXCR7 could regulate several candidate genes related to the PPAR, WNT, Hedgehog or Notch pathways, and their signaling activities, which are known to control cell differentiation and immunomodulation. Finally, MSCs with CXCR7 gain-of-function significantly reduced the articular index scores, ankle circumference, radiographic scores, histologic scores, and inflammation in rats with CIA compared with control MSCs. CONCLUSIONS: CXCR7 promotes the osteogenic and chondrogenic differentiation of MSCs and MSC-mediated immunomodulation by regulating several signaling pathways and anti-inflammatory soluble factors. MSCs with CXCR7 gain-of-function significantly ameliorate arthritic symptoms in a CIA model.

Assessing Age-Related Gray Matter Differences in Young Adults with Voxel-Based Morphometry: The Effect of Field Strengths.

Knowing the patterns of brain differences with age in the young population could lead to a better understanding of the causes of certain psychiatric disorders; however, relevant information is insufficient. Here, a pattern of regional gray matter (GM) that changed with age in a young cohort aged 20-30 years was provided. Extending from previous age studies, all participants were imaged at both 1.5 T and 3 T to address the question of how far the field strength influences results. Fifty-nine young participants aged 20-30 years were scanned at both 1.5 T and 3 T. Voxel-based morphometry (VBM) was used to estimate the GM volume. Some brain regions showed a significant field strength-dependent difference in GM volume. VBM uncovered a significantly age-related increase in the GM volume in the left visual-associated area at 3 T, which was not detected at 1.5 T. In addition, voxels at 1.5 T that revealed a significant age-related reduction in the GM volume were found in the right cerebellum. In conclusion, age-related differences in human brain morphology could even be detected in a young cohort aged 20-30 years; however, the results varied across field strengths. Thus, field strength should be considered an important factor when comparing age-specific brain differences across studies.

Atypical chemokine receptor ACKR3/CXCR7 controls postnatal vasculogenesis and arterial specification by mesenchymal stem cells via Notch signaling.

Mesenchymal stem cells (MSCs) are known to play a role in postnatal vasculogenesis and hold great promise for vascular regeneration. However, the mechanisms by which the endothelial differentiation and specification of MSCs remain unclear. We examined the potential role and molecular mechanisms of atypical chemokine receptor ACKR3/CXCR7 in MSC-mediated endothelial cell differentiation and specification. Here, we showed that vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) activate CXCR7 expression on MSCs through PDGF receptors, PDGFRα and PDGFRß-mediated phosphoinositide 3-kinase (PI3K)/Akt signaling. Genetic and pharmacologic blockage of CXCR7 on MSCs suppressed the VEGF or stromal cell-derived factor 1 (SDF)-1-induced the capacity for vasculogenesis in vitro and in vivo. Moreover, CXCR7 gain of function markedly promoted vasculogenesis by MSCs in vitro and in vivo and induced endothelial differentiation along the arterial endothelial cell lineage via upregulation of Notch signaling. However, blockade of Notch signaling inhibited CXCR7-induced vasculogensis by MSCs. These results indicate CXCR7 is a critical regulator of MSC-mediated postnatal vasculogenesis and arterial specification via Notch signaling.

The effect of linker DNA on the structure and interaction of nucleosome core particles.

In eukaryotes, the compaction of chromatin fibers composed of nucleosome core particles (NCPs) connected by a linker DNA into chromosomes is highly efficient; however, the underlying folding mechanisms remain elusive. We used small angle X-ray scattering (SAXS) to investigate the influence of linker DNA length on the local structure and the interparticle interactions of the NCPs. In the presence of the linker DNA of 30 bp or less in length, the results suggest partial unwrapping of nucleosomal DNA on the NCP irrespective of the linker DNA length. Moreover, the presence of 15 bp linker DNA alleviated the electrostatic repulsion between the NCPs and prevented the formation of an ordered columnar hexagonal phase, demonstrating that the linker DNA plays an active role in chromatin folding.

MRI-based measurements of whole-brain global cerebral blood flow: Comparison and validation at 1.5T and 3T.

BACKGROUND: Whole-brain global cerebral blood flow (CBF) determined by MRI techniques, calculated using total CBF (TCBF) from phase-contrast MRI (PC-MRI), and brain parenchyma volume (BPV) from T1 -weighted image, have become increasingly popular in many applications. PURPOSE/HYPOTHESIS: To determine if MRI-based measurements of whole-brain global CBF data obtained across different field strengths could be merged, TCBF and BPV data acquired at 1.5T and 3T were compared. STUDY TYPE: Prospective study. POPULATION: Seventeen healthy subjects (eight females, aged 21-29 years old). FIELD STRENGTH/SEQUENCE: Fast spoiled gradient echo (FSPGR) and PC-MRI at both 1.5T and 3T. ASSESSMENT: TCBF and BPV data acquired at 1.5T and 3T were compared. STATISTICAL TESTS: The relationships of TCBF and whole-brain global CBF between two field strengths were examined by using the Pearson correlation coefficient analysis and intraclass correlation coefficient (ICC). RESULTS: Regression analysis revealed a strong correlation between TCBF at two field strengths (R2 = 0.78, P < 0.001), and the ICC was 0.85, suggesting measurements of TCBF at 1.5T were comparable and correlated with those at 3T. There was a significant difference in BPV between field strengths, where the white matter estimate was significantly larger at 1.5T when compared with that at 3T (P < 0.001). When TCBF was further normalized to the brain parenchyma mass to obtain whole-brain global CBF, it only showed a moderate correlation between measurements at the two field strengths (R2 = 0.46, P = 0.003) and lower ICC of 0.66, reflecting the slightly higher interstrength variability in the whole-brain global CBF measurements. DATA CONCLUSION: TCBF measurements could be performed equally well with comparable results at both field strengths, but specific attention should be given when TCBF is further normalized to BPV to obtain whole-brain global CBF. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2018;47:1273-1280.

HIF-1α triggers long-lasting glutamate excitotoxicity via system xc- in cerebral ischaemia-reperfusion.

Hypoxia-inducible factor 1α (HIF-1α) controls many genes involved in physiological and pathological processes. However, its roles in glutamatergic transmission and excitotoxicity are unclear. Here, we proposed that HIF-1α might contribute to glutamate-mediated excitotoxicity during cerebral ischaemia-reperfusion (CIR) and investigated its molecular mechanism. We showed that an HIF-1α conditional knockout mouse displayed an inhibition in CIR-induced elevation of extracellular glutamate and N-methyl-d-aspartate receptor (NMDAR) activation. By gene screening for glutamate transporters in cortical cells, we found that HIF-1α mainly regulates the cystine-glutamate transporter (system xc- ) subunit xCT by directly binding to its promoter; xCT and its function are up-regulated in the ischaemic brains of rodents and humans, and the effects lasted for several days. Genetic deletion of xCT in cortical cells of mice inhibits either oxygen glucose deprivation/reoxygenation (OGDR) or CIR-mediated glutamate excitotoxicity in vitro and in vivo. Pharmaceutical inhibition of system xc- by a clinically approved anti-cancer drug, sorafenib, improves infarct volume and functional outcome in rodents with CIR and its therapeutic window is at least 3 days. Taken together, these findings reveal that HIF-1α plays a role in CIR-induced glutamate excitotoxicity via the long-lasting activation of system xc- -dependent glutamate outflow and suggest that system xc- is a promising therapeutic target with an extended therapeutic window in stroke. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Peptide modified polymer poly (glycerol- dodecanedioate co-fumarate) for efficient control of motor neuron differentiation.

Neural tissue engineering is one of the most promising approaches for healing nerve damage, which bypasses the limits of contemporary conventional treatments. In a previous study, we developed a fibrous scaffold via electrospinning poly (glycerol dodecanedioate) (PGD) and gelatin that mimics the structure of a native extracellular matrix (ECM) for soft tissue engineering application. In this study, fumaric acid (FA) was incorporated into the PGD synthesis process, which produced a PGD derivative referred to as poly (glycerol dodecanedioate co-fumarate) (PGDF). This introduced a new functional group, a double bond, into the polymer thus providing new modification possibilities. Arg-Gly-Asp-Cys (RGDC) and laminin peptides were chosen as biomolecules to modify the fiber and facilitate cell attachment and differentiation efficiency. The release of FA into the medium was quantified to investigate the bioreactivity of the derived scaffolds. In combination with UV crosslinking, the developed PGDF fiber mats were able to withstand degradation processes for up to 2 months, which ensures that neural tissue engineering applications are viable. Cell viability and motor neuron differentiation efficiency were demonstrated to be significantly improved with the addition of FA, RGDC and laminin peptides.

Synthesis of hexahydro-1H-isoindole derivatives from arylacyl bromides via homoallenic bromohydrins.

A procedure has been developed for the concise synthesis of hexahydro-1H-isoindole derivatives starting from phenacyl bromides. The approach employs a sequence involving an initial indium-mediated allenylation reaction of an arylacyl bromide with propargyl bromide. This process is followed by FeBr3-mediated SN2'-type substitution reaction of the formed homoallenic bromohydrin to produce a 2,5-dibromo-4-aryl-1,3-pentadiene, which then is subjected to a sequential, one-pot N-alkylation reaction with N-allyl-N-(p-tosyl)amine and a highly diastereoselective intramolecular Diels-Alder reaction of the formed ene-diene to generate the target hexahydro-1H-isoindole.

Thermal and pH Sensitive Multifunctional Polymer Nanoparticles for Cancer Imaging and Therapy.

In this study, we prepared novel poly(Glycerol malate co-dodecanedioate) (PGMD) NPs containing an imaging/hyperthermia agent (IR820) and a chemotherapeutic agent (doxorubicin, DOX). The PGMD polymer was prepared by thermal condensation. IR820 and DOX loaded PGMD NPs were prepared using the single oil emulsion technique. The size of the NPs measured was around 150 nm. Drug loading efficiency of DOX and IR820 was around 4% and 8%, respectively. An acidic environment (pH=5.0) induced higher DOX release as compared to pH=7.4. DOX release was also enhanced by exposure to laser, which increased the temperature to 42°C. Cytotoxicity of the drug loaded NPs was comparable in MES-SA but was higher in Dx5 cells compared to free drug (p<0.05). The combination of hyperthermia and chemotherapy improved cytotoxicity in both cell lines. The NP formulation significantly improved the plasma half-life of IR820 in mice after tail vein injection.

Electrospun fibrous scaffolds of Poly(glycerol-dodecanedioate) for engineering neural tissues from mouse embryonic stem cells.

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task.  Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named poly(glycerol-dodecanoate) (PGD)(1) by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun fiber scaffolds of poly (glycerol-dodecanedioate) and its gelatin blended polymers for soft tissue engineering.

For tissue engineering applications, biodegradable scaffolds play a vital role in supporting and guiding the seeded cells to form functional tissues by mimicking the structure and function of native extracellular matrices. Previously, we have developed a biodegradable elastomer poly (glycerol-dodecanedioate) (PGD) with mechanical properties suitable for soft tissue engineering. In the study, we found that the PGD and PGD blended with gelatin (PGD/gelatin) were able to be electrospun into fibrous scaffolds, and the diameters of the fibers could be adjusted by controlling the PGD concentration. When using our newly designed electrospinning collector, fibers could be easily harvested and the size of the fiber mat could be flexibly adjusted. The data of Raman spectra also confirmed the esterfication reaction in PGD polymerization and showed no significant structure change after electrospinning. Biocompatibility testing of the PGD and PGD/gelatin, by using human foreskin fibroblasts, indicated that gelatin could enhance cell adhesion and proliferation. Overall, electrospun fibers made from PGD and PGD/gelatin exhibited several advantages including easy synthesis from renewable raw materials, flexible fabrication by using less toxic solvents like ethanol, and good biocompatibility.

Near-infrared dye loaded polymeric nanoparticles for cancer imaging and therapy and cellular response after laser-induced heating.

BACKGROUND: In the past decade, researchers have focused on developing new biomaterials for cancer therapy that combine imaging and therapeutic agents. In our study, we use a new biocompatible and biodegradable polymer, termed poly(glycerol malate co-dodecanedioate) (PGMD), for the synthesis of nanoparticles (NPs) and loading of near-infrared (NIR) dyes. IR820 was chosen for the purpose of imaging and hyperthermia (HT). HT is currently used in clinical trials for cancer therapy in combination with radiotherapy and chemotherapy. One of the potential problems of HT is that it can up-regulate hypoxia-inducible factor-1 (HIF-1) expression and enhance vascular endothelial growth factor (VEGF) secretion. RESULTS: We explored cellular response after rapid, short-term and low thermal dose laser-IR820-PGMD NPs (laser/NPs) induced-heating, and compared it to slow, long-term and high thermal dose heating by a cell incubator. The expression levels of the reactive oxygen species (ROS), HIF-1 and VEGF following the two different modes of heating. The cytotoxicity of NPs after laser/NP HT resulted in higher cell killing compared to incubator HT. The ROS level was highly elevated under incubator HT, but remained at the baseline level under the laser/NP HT. Our results show that elevated ROS expression inside the cells could result in the promotion of HIF-1 expression after incubator induced-HT. The VEGF secretion was also significantly enhanced compared to laser/NP HT, possibly due to the promotion of HIF-1. In vitro cell imaging and in vivo healthy mice imaging showed that IR820-PGMD NPs can be used for optical imaging. CONCLUSION: IR820-PGMD NPs were developed and used for both imaging and therapy purposes. Rapid and short-term laser/NP HT, with a low thermal dose, does not up-regulate HIF-1 and VEGF expression, whereas slow and long term incubator HT, with a high thermal dose, enhances the expression of both transcription factors.

γ-Halo-enones: a method for their synthesis from arylacyl halides and their application to the preparation of five-membered ring heterocycles.

A simple method has been developed for synthesis of γ-halo-enones. The approach employs a sequence involving initial indium-mediated allenylation reactions of phenacyl halides with propargyl bromide. This process is followed by acid-promoted rearrangement reactions of the formed homoallenic halohydrins. The new method can be incorporated into routes for the efficient synthesis of various five-membered heterocyclic compounds.

Poly(glycerol-dodecanoate), a biodegradable polyester for medical devices and tissue engineering scaffolds.

In this paper we describe the mechanical and biological features of a thermosetting polyester synthesized from glycerol and dodecanedioic acid named Poly-Glycerol-Dodecanoate (PGD). This polymer shows a glass transition temperature (T(g)) around 32 degrees C, and this accounts for its mechanical properties. At room temperature (21 degrees ) PGD behaves like a stiff elastic-plastic material, while at body temperature (37 degrees C), it shows a compliant non-linear elastic behavior. Together with biodegradability and biocompatibility PGD has distinct shape memory features. After the polymer is cured, no matter what the final configuration is, we can recover the original shape by heating PGD to temperatures of 32 degrees C and higher. The mechanical properties together with biocompatibility/biodegradability and shape memory features make PGD an attractive polymer for biomedical applications.

Cardiac cells implanted into a cylindrical, vascularized chamber in vivo: pressure generation and morphology.

We have previously described a model to implant dissociated cells into a cylindrical, vascularized bed in vivo to promote the formation of functional cardiac muscle constructs. We now investigate the cellular organization and the ability of the constructs to generate intra-luminal pressure. Primary cardiac cells were isolated from hearts of 2-3 day old rats, suspended in fibrin gel and inserted into the lumen of silicone tubing. The silicone tubing was then implanted around the femoral vessels in the groin region of recipient animals. After 3 weeks, the constructs were harvested, placed in an in vitro bath and cannulated via the incorporated femoral artery with a pressure transducer for evaluation of intra-luminal pressure dynamics. Histological evaluation showed the presence of a concentric ring of cardiac cells surrounding the femoral vessels. There was also a significant amount of collagen present around cardiac cells. In addition, we observed a significant amount of neovascularization of the explanted constructs. Electron microscopy showed the presence of longitudinally aligned fibers with a large number of gap junctions. Upon electrical stimulation of a single pulse (7 V, 1.2 ms), the constructs generated an intra-luminal pressure of 1.19 +/- 0.45 mmHg (n = 6). In addition, we were able to electrically pace the constructs at frequencies of 0.5-5 Hz. A Starling behavior of the inverse relation between baseline pressure and twitch pressure was observed. Cardiac cells implanted for 3 weeks into the cylindrical vascularized bed formed a tissue construct that demonstrated many of the contractile properties and morphology expected of functioning cardiac tissues.