Biofabrication of spheroids fusion-based tumor models: computational simulation of glucose effects.

In vitrotumor models consisting of cell spheroids are increasingly used for mechanistic studies and pharmacological testing. However, unless vascularized, the availability of nutrients such as glucose to deeper layers of multicellular aggregates is limited. In addition, recent developments in cells-only biofabrication (e.g. 'scaffold-free bioprinting'), allow the creation of more complex spheroid-based structures, further exposing the cells to nutrient deprivation within these constructs. To explore the impact of glucose availability on such tumor-like structures, we used the CompuCell3D platform for modeling of tumor spheroids. By monitoring the types of cells, fusing pairs geometry and the distance between spheroids centers of mass, we made novel heuristic observations on how binary- and multi-spheroid fusions are impacted by glucose availability. At limiting glucose concentrations mimicking hypoglycemia we noted an abrupt collapse of the tumor spheroids, unexpectedly amplified by the contact with normal cell spheroids. At higher glucose concentrations, we found an increased intermixing of cancerous cells, strong anti-phase oscillations between proliferating and quiescent tumor cells and a structural instability of fusing tumor spheroids, leading to their re-fragmentation. In a model of tumor microenvironment composed of normal cell spheroids fusing around a tumoral one, the competition for glucose lead to either the tumor's disappearance, to a steady state, or to its expansion. Moreover, the invasion of this microenvironment by individual tumor cells was also strongly depended on the available glucose. In conclusion, we demonstrate the value of computational simulations for anticipating the properties of biofabricated tumor models, and in generating testable hypotheses regarding the relationship between cancer, nutrition and diabetes.

Three-Dimensional Bioprinting of Anatomically Realistic Tissue Constructs for Disease Modeling and Drug Testing.

Three-dimensional (3D) bioprinting is an emerging tissue engineering technology, already with several remarkable accomplishments and with more promises to fulfill. Besides the enduring goal of making tissues for implantation, it could also become an essential tool in the worldwide trend to replace animal experimentation with improved in vitro models for disease mechanism studies, or with new high-throughput pharmacological and toxicology assays. All these require the speed, reproducibility, and standardization that bioprinting could easily provide. However, originating from additive manufacturing with its top-down approach of "filling" a virtual volume with a semifluid (hydrogel) material, the finer internal anatomic structure of the tissues, as well as vascularization and innervation, has remained difficult to implement. Thus, the next frontier in bioprinting is the generation of more anatomically realistic models, needed for ascending to the functionality of living tissues. In this study, I discuss the conceptual and practical barriers still hampering the attainment of this goal and suggest solutions to overcome them. In this regard, I introduce two workflows that combine existing methods in new operational sequences: (1) bioprinting guided by images of histological sections assembled in 3D constructs and (2) bioprinting of bidimensional vascular patterns implemented among stackable cellular layers. While more sophisticated methods to capture the tissue structure in 3D constructs certainly exist, I contend that extrusion bioprinting may still offer a simple, practical, and affordable option. Impact statement Paucity of anatomic structural details is one of the limitations of three-dimensional bioprinting toward fulfilling its potential for tissue engineering, drug testing, and toxicological assays. The origins of this problem can be tracked back to derivation of bioprinting from inorganic additive manufacturing, making it more adept to render the shapes of the objects than their content. As solutions, I suggest two simple workflows that can be implemented by most current bioprinters, based on the import into the construct design of anatomically realistic structural information. If more largely adopted, these and similar approaches may significantly improve the applicability of bioprinted constructs.

Of balls, inks and cages: Hybrid biofabrication of 3D tissue analogs.

The overarching principle of three-dimensional (3D) bioprinting is the placing of cells or cell clusters in the 3D space to generate a cohesive tissue microarchitecture that comes close to in vivo characteristics. To achieve this goal, several technical solutions are available, generating considerable combinatorial bandwidth: (i) Support structures are generated first, and cells are seeded subsequently; (ii) alternatively, cells are delivered in a printing medium, so-called "bioink," that contains them during the printing process and ensures shape fidelity of the generated structure; and (iii) a "scaffold-free" version of bioprinting, where only cells are used and the extracellular matrix is produced by the cells themselves, also recently entered a phase of accelerated development and successful applications. However, the scaffold-free approaches may still benefit from secondary incorporation of scaffolding materials, thus expanding their versatility. Reversibly, the bioink-based bioprinting could also be improved by adopting some of the principles and practices of scaffold-free biofabrication. Collectively, we anticipate that combinations of these complementary methods in a "hybrid" approach, rather than their development in separate technological niches, will largely increase their efficiency and applicability in tissue engineering.

Labeling of endothelial cells with magnetic microbeads by angiophagy.

OBJECTIVES: Attachment of magnetic particles to cells is needed for a variety of applications but is not always possible or efficient. Simpler and more convenient methods are thus desirable. In this study, we tested the hypothesis that endothelial cells (EC) can be loaded with micron-size magnetic beads by the phagocytosis-like mechanism 'angiophagy'. To this end, human umbilical vein EC (HUVEC) were incubated with magnetic beads conjugated or not (control) with an anti-VEGF receptor 2 antibody, either in suspension, or in culture followed by re-suspension using trypsinization. RESULTS: In all conditions tested, HUVEC incubation with beads induced their uptake by angiophagy, which was confirmed by (i) increased cell granularity assessed by flow cytometry, and (ii) the presence of an F-actin rich layer around many of the intracellular beads, visualized by confocal microscopy. For confluent cultures, the average number of beads per cell was 4.4 and 4.2, with and without the presence of the anti-VEGFR2 antibody, respectively. However, while the actively dividing cells took up 2.9 unconjugated beads on average, this number increased to 5.2 if binding was mediated by the antibody. Magnetic pulldown increased the cell density of beads-loaded cells in porous electrospun poly-capro-lactone scaffolds by a factor of 4.5 after 5 min, as compared to gravitational settling (p < 0.0001). CONCLUSION: We demonstrated that EC can be readily loaded by angiophagy with micron-sized beads while attached in monolayer culture, then dispersed in single-cell suspensions for pulldown in porous scaffolds and for other applications.

Progress in scaffold-free bioprinting for cardiovascular medicine.

Biofabrication of tissue analogues is aspiring to become a disruptive technology capable to solve standing biomedical problems, from generation of improved tissue models for drug testing to alleviation of the shortage of organs for transplantation. Arguably, the most powerful tool of this revolution is bioprinting, understood as the assembling of cells with biomaterials in three-dimensional structures. It is less appreciated, however, that bioprinting is not a uniform methodology, but comprises a variety of approaches. These can be broadly classified in two categories, based on the use or not of supporting biomaterials (known as "scaffolds," usually printable hydrogels also called "bioinks"). Importantly, several limitations of scaffold-dependent bioprinting can be avoided by the "scaffold-free" methods. In this overview, we comparatively present these approaches and highlight the rapidly evolving scaffold-free bioprinting, as applied to cardiovascular tissue engineering.

iPSC-Derived Vascular Cell Spheroids as Building Blocks for Scaffold-Free Biofabrication.

Recently a protocol is established to obtain large quantities of human induced pluripotent stem cells (iPSC)-derived endothelial progenitors, called endothelial colony forming cells (ECFC), and of candidate smooth-muscle forming cells (SMFC). Here, the suitability for assembling in spheroids, and in larger 3D cell constructs is tested. iPSC-derived ECFC and SMFC are labeled with tdTomato and eGFP, respectively. Spheroids are formed in ultra-low adhesive wells, and their dynamic proprieties are studied by time-lapse microscopy, or by confocal microscopy. Spheroids are also tested for fusion ability either in the wells, or assembled on the Regenova 3D bioprinter which laces them in stainless steel micro-needles (the "Kenzan" method). It is found that both ECFC and SMFC formed spheroids in about 24 h. Fluorescence monitoring indicated a continuous compaction of ECFC spheroids, but stabilization in those prepared from SMFC. In mixed spheroids, the cell distribution changed continuously, with ECFC relocating to the core, and showing pre-vascular organization. All spheroids have the ability of in-well fusion, but only those containing SMFC are robust enough to sustain assembling in tubular structures. In these constructs a layered distribution of alpha smooth muscle actin-positive cells and extracellular matrix deposition is found. In conclusion, iPSC-derived vascular cell spheroids represent a promising new cellular material for scaffold-free biofabrication.

Mechanotransduction Effects on Endothelial Cell Proliferation via CD31 and VEGFR2: Implications for Immunomagnetic Separation.

Immunomagnetic separation is used to isolate circulating endothelial cells (ECs) and endothelial progenitor cells (EPCs) for diagnostics and tissue engineering. However, potentially detrimental changes in cell properties have been observed post-separation. Here, the effect of mechanical force, which is naturally applied during immunomagnetic separation, on proliferation of human umbilical vein endothelial cells (HUVEC), kinase insert domain-positive receptor (KDR) cells, and peripheral blood mononuclear cells (PBMCs). Cells are exposed to CD31 or Vascular Endothelial Growth Factor Receptor-2 (VEGFR2) targeted MACSi beads at varying bead to cell ratios and compared to free antibody and unconjugated beads. A vertical magnetic gradient is applied to static 2D cultures, and a magnetic cell sorter is used to analyze cells in dynamic flow. No significant difference in EC proliferation is observed for controls or VEGFR2-targeting beads, whereas CD31-conjugated beads increase proliferation in a dose dependent manner in static 2-D cultures. This effect occurs in the absence of magnetic field, but is more pronounced with magnetic force. After flow sorting, similar increases in proliferation are seen for CD31 targeting beads. Thus, the effects of targeting antibody and magnetic force applied should be considered when designing immunomagnetic separation protocols for ECs.

Transcriptional Networks in Single Perivascular Cells Sorted from Human Adipose Tissue Reveal a Hierarchy of Mesenchymal Stem Cells.

Adipose tissue is a rich source of multipotent mesenchymal stem-like cells, located in the perivascular niche. Based on their surface markers, these have been assigned to two main categories: CD31- /CD45- /CD34+ /CD146- cells (adventitial stromal/stem cells [ASCs]) and CD31- /CD45- /CD34- /CD146+ cells (pericytes [PCs]). These populations display heterogeneity of unknown significance. We hypothesized that aldehyde dehydrogenase (ALDH) activity, a functional marker of primitivity, could help to better define ASC and PC subclasses. To this end, the stromal vascular fraction from a human lipoaspirate was simultaneously stained with fluorescent antibodies to CD31, CD45, CD34, and CD146 antigens and the ALDH substrate Aldefluor, then sorted by fluorescence-activated cell sorting. Individual ASCs (n = 67) and PCs (n = 73) selected from the extremities of the ALDH-staining spectrum were transcriptionally profiled by Fluidigm single-cell quantitative polymerase chain reaction for a predefined set (n = 429) of marker genes. To these single-cell data, we applied differential expression and principal component and clustering analysis, as well as an original gene coexpression network reconstruction algorithm. Despite the stochasticity at the single-cell level, covariation of gene expression analysis yielded multiple network connectivity parameters suggesting that these perivascular progenitor cell subclasses possess the following order of maturity: (a) ALDHbr ASC (most primitive); (b) ALDHdim ASC; (c) ALDHbr PC; (d) ALDHdim PC (least primitive). This order was independently supported by specific combinations of class-specific expressed genes and further confirmed by the analysis of associated signaling pathways. In conclusion, single-cell transcriptional analysis of four populations isolated from fat by surface markers and enzyme activity suggests a developmental hierarchy among perivascular mesenchymal stem cells supported by markers and coexpression networks. Stem Cells 2017;35:1273-1289.

Principles of the Kenzan Method for Robotic Cell Spheroid-Based Three-Dimensional Bioprinting.

Bioprinting is a technology with the prospect to change the way many diseases are treated, by replacing the damaged tissues with live de novo created biosimilar constructs. However, after more than a decade of incubation and many proofs of concept, the field is still in its infancy. The current stagnation is the consequence of its early success: the first bioprinters, and most of those that followed, were modified versions of the three-dimensional printers used in additive manufacturing, redesigned for layer-by-layer dispersion of biomaterials. In all variants (inkjet, microextrusion, or laser assisted), this approach is material ("scaffold") dependent and energy intensive, making it hardly compatible with some of the intended biological applications. Instead, the future of bioprinting may benefit from the use of gentler scaffold-free bioassembling methods. A substantial body of evidence has accumulated, indicating this is possible by use of preformed cell spheroids, which have been assembled in cartilage, bone, and cardiac muscle-like constructs. However, a commercial instrument capable to directly and precisely "print" spheroids has not been available until the invention of the microneedles-based ("Kenzan") spheroid assembling and the launching in Japan of a bioprinter based on this method. This robotic platform laces spheroids into predesigned contiguous structures with micron-level precision, using stainless steel microneedles ("kenzans") as temporary support. These constructs are further cultivated until the spheroids fuse into cellular aggregates and synthesize their own extracellular matrix, thus attaining the needed structural organization and robustness. This novel technology opens wide opportunities for bioengineering of tissues and organs.

Actin grips: circular actin-rich cytoskeletal structures that mediate the wrapping of polymeric microfibers by endothelial cells.

Interaction of endothelial-lineage cells with three-dimensional substrates was much less studied than that with flat culture surfaces. We investigated the in vitro attachment of both mature endothelial cells (ECs) and of less differentiated EC colony-forming cells to poly-ε-capro-lactone (PCL) fibers with diameters in 5-20 µm range ('scaffold microfibers', SMFs). We found that notwithstanding the poor intrinsic adhesiveness to PCL, both cell types completely wrapped the SMFs after long-term cultivation, thus attaining a cylindrical morphology. In this system, both EC types grew vigorously for more than a week and became increasingly more differentiated, as shown by multiplexed gene expression. Three-dimensional reconstructions from multiphoton confocal microscopy images using custom software showed that the filamentous (F) actin bundles took a conspicuous ring-like organization around the SMFs. Unlike the classical F-actin-containing stress fibers, these rings were not associated with either focal adhesions or intermediate filaments. We also demonstrated that plasma membrane boundaries adjacent to these circular cytoskeletal structures were tightly yet dynamically apposed to the SMFs, for which reason we suggest to call them 'actin grips'. In conclusion, we describe a particular form of F-actin assembly with relevance for cytoskeletal organization in response to biomaterials, for endothelial-specific cell behavior in vitro and in vivo, and for tissue engineering.

A module of human peripheral blood mononuclear cell transcriptional network containing primitive and differentiation markers is related to specific cardiovascular health variables.

Peripheral blood mononuclear cells (PBMCs), including rare circulating stem and progenitor cells (CSPCs), have important yet poorly understood roles in the maintenance and repair of blood vessels and perfused organs. Our hypothesis was that the identities and functions of CSPCs in cardiovascular health could be ascertained by analyzing the patterns of their co-expressed markers in unselected PBMC samples. Because gene microarrays had failed to detect many stem cell-associated genes, we performed quantitative real-time PCR to measure the expression of 45 primitive and tissue differentiation markers in PBMCs from healthy and hypertensive human subjects. We compared these expression levels to the subjects' demographic and cardiovascular risk factors, including vascular stiffness. The tested marker genes were expressed in all of samples and organized in hierarchical transcriptional network modules, constructed by a bottom-up approach. An index of gene expression in one of these modules (metagene), defined as the average standardized relative copy numbers of 15 pluripotency and cardiovascular differentiation markers, was negatively correlated (all p<0.03) with age (R2 = -0.23), vascular stiffness (R2 = -0.24), and central aortic pressure (R2 = -0.19) and positively correlated with body mass index (R2 = 0.72, in women). The co-expression of three neovascular markers was validated at the single-cell level using mRNA in situ hybridization and immunocytochemistry. The overall gene expression in this cardiovascular module was reduced by 72±22% in the patients compared with controls. However, the compactness of both modules was increased in the patients' samples, which was reflected in reduced dispersion of their nodes' degrees of connectivity, suggesting a more primitive character of the patients' CSPCs. In conclusion, our results show that the relationship between CSPCs and vascular function is encoded in modules of the PBMCs transcriptional network. Furthermore, the coordinated gene expression in these modules can be linked to cardiovascular risk factors and subclinical cardiovascular disease; thus, this measure may be useful for their diagnosis and prognosis.

Reoxygenation-derived toxic reactive oxygen/nitrogen species modulate the contribution of bone marrow progenitor cells to remodeling after myocardial infarction.

BACKGROUND: The core region of a myocardial infarction is notoriously unsupportive of cardiomyocyte survival. However, there has been less investigation of the potentially beneficial spontaneous recruitment of endogenous bone marrow progenitor cells (BMPCs) within infarcted areas. In the current study we examined the role of tissue oxygenation and derived toxic species in the control of BMPC engraftment during postinfarction heart remodeling. METHODS AND RESULTS: For assessment of cellular origin, local oxygenation, redox status, and fate of cells in the infarcted region, myocardial infarction in mice with or without LacZ(+) bone marrow transplantation was induced by coronary ligation. Sham-operated mice served as controls. After 1 week, LacZ(+) BMPC-derived cells were found inhomogeneously distributed into the infarct zone, with a lower density at its core. Electron paramagnetic resonance (EPR) oximetry showed that pO2 in the infarct recovered starting on day 2 post-myocardial infarction, concomitant with wall thinning and erythrocytes percolating through muscle microruptures. Paralleling this reoxygenation, increased generation of reactive oxygen/nitrogen species was detected at the infarct core. This process delineated a zone of diminished BMPC engraftment, and at 1 week infiltrating cells displayed immunoreactive 3-nitrotyrosine and apoptosis. In vivo treatment with a superoxide dismutase mimetic significantly reduced reactive oxygen species formation and amplified BMPC accumulation. This treatment also salvaged wall thickness by 43% and left ventricular ejection fraction by 27%, with significantly increased animal survival. CONCLUSIONS: BMPC engraftment in the infarct inversely mirrored the distribution of reactive oxygen/nitrogen species. Antioxidant treatment resulted in increased numbers of engrafted BMPCs, provided functional protection to the heart, and decreased the incidence of myocardial rupture and death.

In vitro endothelialization of electrospun terpolymer scaffolds: evaluation of scaffold type and cell source.

A family of methacrylic terpolymer biomaterials was electrospun into three-dimensional scaffolds. The glass transition temperature of the polymer correlates with the morphology of the resulting scaffold. Glassy materials produce scaffolds with discrete fibers and large pore areas (1531±1365 µm(2)), while rubbery materials produce scaffolds with fused fibers and smaller pore areas (154±110 µm(2)). Three different endothelial-like cell populations were seeded onto these scaffolds under static conditions: human umbilical vein endothelial cells (HUVECs), adult human peripheral blood-derived outgrowth endothelial cells, and umbilical cord blood-derived human blood outgrowth endothelial cells. Cellular behavior depended on both cell type and scaffold topography. Specifically, cord blood-derived outgrowth endothelial cells showed more robust adhesion and growth on all scaffolds in comparison to other cell types as measured by the density of adherent cells, the number of proliferative cells, and the enzymatic activity of the adherent cells. Peripheral blood-derived outgrowth cells exhibited less ability to inhabit the terpolymer interfaces in comparison to their cord blood-derived counterparts. HUVECs also exhibited less of a capacity to colonize the terpolymer interfaces in comparison to the cord blood-derived cells. However, the mature endothelial cells did show scaffold-dependent behavior. Specifically, we observed an increase in their ability to populate the low-porosity scaffolds. All cells maintained an endothelial phenotype after 1 week of culture on the electrospun scaffolds.

Modeling stem/progenitor cell-induced neovascularization and oxygenation around solid implants.

Tissue engineering constructs and other solid implants with biomedical applications, such as drug delivery devices or bioartificial organs, need oxygen (O(2)) to function properly. To understand better the vascular integration of such devices, we recently developed a novel model sensor containing O(2)-sensitive crystals, consisting of a polymeric capsule limited by a nanoporous filter. The sensor was implanted in mice with hydrogel alone (control) or hydrogel embedded with mouse CD117/c-kit+ bone marrow progenitor cells in order to stimulate peri-implant neovascularization. The sensor provided local partial O(2) pressure (pO(2)) using noninvasive electron paramagnetic resonance signal measurements. A consistently higher level of peri-implant oxygenation was observed in the cell-treatment case than in the control over a 10-week period. To provide a mechanistic explanation of these experimental observations, we present in this article a mathematical model, formulated as a system of coupled partial differential equations, that simulates peri-implant vascularization. In the control case, vascularization is considered to be the result of a foreign body reaction, while in the cell-treatment case, adipogenesis in response to paracrine stimuli produced by the stem cells is assumed to induce neovascularization. The model is validated by fitting numerical predictions of local pO(2) to measurements from the implanted sensor. The model is then used to investigate further the potential for using stem cell treatment to enhance the vascular integration of biomedical implants. We thus demonstrate how mathematical modeling combined with experimentation can be used to infer how vasculature develops around biomedical implants in control and stem cell-treated cases.

Dynamics of the cytoskeleton: how much does water matter?

The principal constituent of the living cell is water. The role of the hydration shell and bulk H(2)O solvent is well recognized in the dynamics of isolated proteins, but the role of water in the dynamics of the integrated living cytoskeleton (CSK) remains obscure. Here we report a direct connection of dynamics of water to dynamics of the integrated CSK. The latter are known to be scale-free and to hinge upon a frequency f(0) that is roughly invariant across cell types. Although f(0) is comparable in magnitude to the rotational relaxation frequency of water (gigahertz range), the physical basis of f(0) remains unknown. Using the human airway smooth muscle cell as a model system, we show here that replacing water acutely with deuterium oxide impacts CSK dynamics in major ways, slowing CSK remodeling dynamics appreciably, and lowering f(0) by up to four orders of magnitude. Although these observations do not distinguish contributions of bulk solvent versus hydration shell, they suggest a unifying hypothesis, namely, that dynamics of integrated CSK networks are slaved in a direct fashion to fluctuations arising in intracellular water.

The stimulation of the cardiac differentiation of mesenchymal stem cells in tissue constructs that mimic myocardium structure and biomechanics.

We investigated whether tissue constructs resembling structural and mechanical properties of the myocardium would induce mesenchymal stem cells (MSCs) to differentiate into a cardiac lineage, and whether further mimicking the 3-D cell alignment of myocardium would enhance cardiac differentiation. The tissue constructs were generated by integrating MSCs with elastic polyurethane nanofibers in an electrical field. Control of processing parameters resulted in tissue constructs recapitulating the fibrous and anisotropic structure, and typical stress-strain response of native porcine myocardium. MSCs proliferated in the tissue constructs when cultured dynamically, but retained a round morphology. mRNA expression demonstrated that cardiac differentiation was significantly stimulated. Enhanced cardiac differentiation was achieved by 3-D alignment of MSCs within the tissue constructs. Cell alignment was attained by statically stretching tissue constructs during culture. Increasing stretching strain from 25% to 75% increased the degree of 3-D cell alignment. Real time RT-PCR results showed that when cells assuming a high degree of alignment (with application of 75% strain), their expression of cardiac markers (GATA4, Nkx2.5 and MEF2C) remarkably increased. The differentiated cells also developed calcium channels, which are required to have electrophysiological properties. This report to some extent explains the outcome of many in vivo studies, where only a limited amount of the injected MSCs differentiated into cardiomyocytes. It is possible that the strain of the heartbeat (∼20%) cannot allow the MSCs to have an alignment high enough for a remarkable cardiac differentiation. This work suggests that pre-differentiation of MSCs into cardiomyocytes prior to injection may result in a greater degree of cardiac regeneration than simply injecting un-differentiated MSCs into heart.

Role of heat shock factor-1 activation in the doxorubicin-induced heart failure in mice.

Treating cancer patients with chemotherapeutics, such as doxorubicin (Dox), cause dilated cardiomyopathy and congestive heart failure because of oxidative stress. On the other hand, heat shock factor-1 (HSF-1), a transcription factor for heat shock proteins (Hsps), is also known to be activated in response to oxidative stress. However, the possible role of HSF-1 activation and the resultant Hsp25 in chemotherapeutic-induced heart failure has not been investigated. Using HSF-1 wild-type (HSF-1(+/+)) and knock-out (HSF-1(-/-)) mice, we tested the hypothesis that activation of HSF-1 plays a role in the development of Dox-induced heart failure. Higher levels of Hsp25 and its phosphorylated forms were found in the failing hearts of Dox-treated HSF-1(+/+) mice. More than twofold increase in Hsp25 mRNA level was found in Dox-treated hearts. Proteomic analysis showed that there is accumulation and aggregation of Hsp25 in Dox-treated failing hearts. Additionally, Hsp25 was found to coimmunoprecipitate with p53 and vice versa. Further studies indicated that the Dox-induced higher levels of Hsp25 transactivated p53 leading to higher levels of the pro-apoptotic protein Bax, but other p53-related proteins remained unaltered. Moreover, HSF-1(-/-) mice showed significantly reduced Dox-induced heart failure and higher survival rate, and there was no change in Bax upon treating with Dox in HSF-1(-/-) mice. From these results we propose a novel mechanism for Dox-induced heart failure: increased expression of Hsp25 because of oxidant-induced activation of HSF-1 transactivates p53 to increase Bax levels, which leads to heart failure.

EPCs and pathological angiogenesis: when good cells go bad.

Bone-marrow-derived endothelial progenitor cells (EPCs) contribute to angiogenesis-mediated pathological neovascularization, and recent studies have begun to recognize the biological significance of this contribution. This review will discuss the ability of EPCs to contribute to neovascularization in both physiological and pathological conditions. Circulating EPCs were originally identified in 1997 by Asahara as CD34(+) VEGFR2(+) mononuclear cells. These cells differentiated into an endothelial phenotype, expressed endothelial markers, and incorporated into neovessels at sites of ischemia (Asahara et al., 1997). EPCs provide both instructive (release of pro-angiogenic cytokines) and structural (vessel incorporation and stabilization) functions that contribute to the initiation of neo-angiogenesis. EPC populations can be characterized based on surface markers of freshly isolated cells, or they can be described by their in vitro characteristics once placed in culture. However, a major stumbling block to progress in the field has been the lack of consensus among investigators as to the optimal characterization of EPCs. This review intends to address the role of both EPC classes and evaluate how they interact in the setting of pathological angiogenesis. Since the EPCs may be responsible for turning on the "angiogenic switch," strategies have been employed to keep this switch in the "off" position for diseases like cancer, retinopathy, and wet AMD. The expectation is that EPCs will evolve into clinically useful prognostic and predictive tools in cancer and in ocular diseases associated with pathological neovascularization and that targeting this cell type is a key to successful management of patients suffering from diseases associated with pathological neovascularization.

Regulation of adult hematopoietic stem cells fate for enhanced tissue-specific repair.

The ability to control the differentiation of adult hematopoietic stem cells (HSCs) would promote development of new cell-based therapies to treat multiple degenerative diseases. Systemic injection of NaIO(3) was used to ablate the retinal pigment epithelial (RPE) layer in C57Bl6 mice and initiate neural retinal degeneration. HSCs infected ex vivo with lentiviral vector expressing the RPE-specific gene RPE65 restored a functional RPE layer, with typical RPE phenotype including coexpression of another RPE-specific marker, CRALBP, and photoreceptor outer segment phagocytosis. Retinal degeneration was prevented and visual function, as measured by electroretinography (ERG), was restored to levels similar to that found in normal animals. None of the controls (no HSCs, HSCs alone and HSCs infected with lentiviral vector expressing LacZ) showed these effects. In vitro gene array studies demonstrated that infection of HSC with RPE65 increased adenylate cyclase mRNA. In vitro exposure of HSCs to a pharmacological agonist of adenylate cyclase also led to in vitro differentiation of HSCs to RPE-like cells expressing pigment granules and the RPE-specific marker, CRALBP. Our data confirm that expression of the cell-specific gene RPE65 promoted fate determination of HSCs toward RPE for targeted tissue repair, and did so in part by activation of adenylate cyclase signaling pathways. Expression by HSCs of single genes unique to a differentiated cell may represent a novel experimental paradigm to influence HSC plasticity, force selective differentiation, and ultimately lead to identification of pharmacological alternatives to viral gene delivery.