Hypoxia-Inducible Factor α Subunits Regulate Tie2-Expressing Macrophages That Influence Tumor Oxygen and Perfusion in Murine Breast Cancer.

Tie2-expressing monocytes/macrophages (TEMs) are a distinct subset of proangiogenic monocytes selectively recruited to tumors in breast cancer. Because of the hypoxic nature of solid tumors, we investigated if oxygen, via hypoxia-inducible transcription factors HIF-1α and HIF-2α, regulates TEM function in the hypoxic tumor microenvironment. We orthotopically implanted PyMT breast tumor cells into the mammary fat pads of syngeneic LysMcre, HIF-1α fl/fl /LysMcre, or HIF-2α fl/fl /LysMcre mice and evaluated the tumor TEM population. There was no difference in the percentage of tumor macrophages among the mouse groups. In contrast, HIF-1α fl/fl /LysMcre mice had a significantly smaller percentage of tumor TEMs compared with control and HIF-2α fl/fl /LysMcre mice. Proangiogenic TEMs in macrophage HIF-2α-deficient tumors presented significantly more CD31+ microvessel density but exacerbated hypoxia and tissue necrosis. Reduced numbers of proangiogenic TEMs in macrophage HIF-1α-deficient tumors presented significantly less microvessel density but tumor vessels that were more functional as lectin injection revealed more perfusion, and functional electron paramagnetic resonance analysis revealed more oxygen in those tumors. Macrophage HIF-1α-deficient tumors also responded significantly to chemotherapy. These data introduce a previously undescribed and counterintuitive prohypoxia role for proangiogenic TEMs in breast cancer which is, in part, suppressed by HIF-2α.

Loss of Angiotensin-Converting Enzyme 2 Exacerbates Diabetic Retinopathy by Promoting Bone Marrow Dysfunction.

Angiotensin-converting enzyme 2 (ACE2) is the primary enzyme of the vasoprotective axis of the renin angiotensin system (RAS). We tested the hypothesis that loss of ACE2 would exacerbate diabetic retinopathy by promoting bone marrow dysfunction. ACE2-/y were crossed with Akita mice, a model of type 1 diabetes. When comparing the bone marrow of the ACE2-/y -Akita mice to that of Akita mice, we observed a reduction of both short-term and long-term repopulating hematopoietic stem cells, a shift of hematopoiesis toward myelopoiesis, and an impairment of lineage- c-kit+ hematopoietic stem/progenitor cell (HS/PC) migration and proliferation. Migratory and proliferative dysfunction of these cells was corrected by exposure to angiotensin-1-7 (Ang-1-7), the protective peptide generated by ACE2. Over the duration of diabetes examined, ACE2 deficiency led to progressive reduction in electrical responses assessed by electroretinography and to increases in neural infarcts observed by fundus photography. Compared with Akita mice, ACE2-/y -Akita at 9-months of diabetes showed an increased number of acellular capillaries indicative of more severe diabetic retinopathy. In diabetic and control human subjects, CD34+ cells, a key bone marrow HS/PC population, were assessed for changes in mRNA levels for MAS, the receptor for Ang-1-7. Levels were highest in CD34+ cells from diabetics without retinopathy. Higher serum Ang-1-7 levels predicted protection from development of retinopathy in diabetics. Treatment with Ang-1-7 or alamandine restored the impaired migration function of CD34+ cells from subjects with retinopathy. These data support that activation of the protective RAS within HS/PCs may represents a therapeutic strategy for prevention of diabetic retinopathy. Stem Cells 2018;36:1430-1440.

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.

Electroacupuncture Promotes Central Nervous System-Dependent Release of Mesenchymal Stem Cells.

Electroacupuncture (EA) performed in rats and humans using limb acupuncture sites, LI-4 and LI-11, and GV-14 and GV-20 (humans) and Bai-hui (rats) increased functional connectivity between the anterior hypothalamus and the amygdala and mobilized mesenchymal stem cells (MSCs) into the systemic circulation. In human subjects, the source of the MSC was found to be primarily adipose tissue, whereas in rodents the tissue sources were considered more heterogeneous. Pharmacological disinhibition of rat hypothalamus enhanced sympathetic nervous system (SNS) activation and similarly resulted in a release of MSC into the circulation. EA-mediated SNS activation was further supported by browning of white adipose tissue in rats. EA treatment of rats undergoing partial rupture of the Achilles tendon resulted in reduced mechanical hyperalgesia, increased serum interleukin-10 levels and tendon remodeling, effects blocked in propranolol-treated rodents. To distinguish the afferent role of the peripheral nervous system, phosphoinositide-interacting regulator of transient receptor potential channels (Pirt)-GCaMP3 (genetically encoded calcium sensor) mice were treated with EA acupuncture points, ST-36 and LIV-3, and GV-14 and Bai-hui and resulted in a rapid activation of primary sensory neurons. EA activated sensory ganglia and SNS centers to mediate the release of MSC that can enhance tissue repair, increase anti-inflammatory cytokine production and provide pronounced analgesic relief. Stem Cells 2017;35:1303-1315.

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.

Adeno-Associated Virus Overexpression of Angiotensin-Converting Enzyme-2 Reverses Diabetic Retinopathy in Type 1 Diabetes in Mice.

Angiotensin-converting enzyme (ACE)-2 is the primary enzyme of the vasoprotective axis of the renin angiotensin system that regulates the classic renin angiotensin system axis. We aimed to determine whether local retinal overexpression of adenoassociated virus (AAV)-ACE2 prevents or reverses diabetic retinopathy. Green fluorescent protein (GFP)-chimeric mice were generated to distinguish resident (retinal) from infiltrating bone marrow-derived inflammatory cells and were made diabetic using streptozotocin injections. Retinal digestion using trypsin was performed and acellular capillaries enumerated. Capillary occlusion by GFP(+) cells was used to measure leukostasis. Overexpression of ACE2 prevented (prevention cohort: untreated diabetic, 11.3 ± 1.4; ACE2 diabetic, 6.4 ± 0.9 per mm(2)) and partially reversed (reversal cohort: untreated diabetic, 15.7 ± 1.9; ACE2 diabetic, 6.5 ± 1.2 per mm(2)) the diabetes-associated increase of acellular capillaries and the increase of infiltrating inflammatory cells into the retina (F4/80(+)) (prevention cohort: untreated diabetic, 24.2 ± 6.7; ACE2 diabetic, 2.5 ± 1.6 per mm(2); reversal cohort: untreated diabetic, 56.8 ± 5.2; ACE2 diabetic, 5.6 ± 2.3 per mm(2)). In both study cohorts, intracapillary bone marrow-derived cells, indicative of leukostasis, were only observed in diabetic animals receiving control AAV injections. These results indicate that diabetic retinopathy, and possibly other diabetic microvascular complications, can be prevented and reversed by locally restoring the balance between the classic and vasoprotective renin angiotensin system.

Macrophage microvesicles induce macrophage differentiation and miR-223 transfer.

Microvesicles are small membrane-bound particles comprised of exosomes and various-sized extracellular vesicles. These are released by several cell types. Microvesicles have a variety of cellular functions from communication to mediating growth and differentiation. Microvesicles contain proteins and nucleic acids. Previously, we showed that plasma microvesicles contain microRNAs (miRNAs). Based on our previous report, the majority of peripheral blood microvesicles are derived from platelets, while mononuclear phagocytes, including macrophages, are the second most abundant population. Here, we characterized macrophage-derived microvesicles and explored their role in the differentiation of naive monocytes. We also identified the miRNA content of the macrophage-derived microvesicles. We found that RNA molecules contained in the macrophage-derived microvesicles were transported to target cells, including mono cytes, endothelial cells, epithelial cells, and fibroblasts. Furthermore, we found that miR-223 was transported to target cells and was functionally active. Based on our observations, we hypothesize that microvesicles bind to and activate target cells. Furthermore, we find that microvesicles induce the differentiation of macrophages. Thus, defining key components of this response may identify novel targets to regulate host defense and inflammation.

Methodological challenges in utilizing miRNAs as circulating biomarkers.

MicroRNAs (miRNAs) have emerged as important regulators in the post-transcriptional control of gene expression. The discovery of their presence not only in tissues but also in extratissular fluids, including blood, urine and cerebro-spinal fluid, together with their changes in expression in various pathological conditions, has implicated these extracellular miRNAs as informative biomarkers of disease. However, exploiting miRNAs in this capacity requires methodological rigour. Here, we report several key procedural aspects of miRNA isolation from plasma and serum, as exemplified by research in cardiovascular and pulmonary diseases. We also highlight the advantages and disadvantages of various profiling methods to determine the expression levels of plasma- and serum-derived miRNAs. Attention to such methodological details is critical, as circulating miRNAs become diagnostic tools for various human diseases.

Myocardial Rac1 exhibits partial involvement in thyroxin-induced cardiomyocyte hypertrophy and its inhibition is not sufficient to improve cardiac dysfunction or contractile abnormalities in mouse papillary muscles.

: Development of cardiac hypertrophy after thyroxin (T4) treatment is well recognized. Recently, we observed that T4-induced cardiac hypertrophy is associated with increased cardiac Rac1 expression and activity. Whether this Rac1 increase has a role in inducing this cardiac phenotype is, however, still unknown. Here, we showed that T4 treatment (500 µg/kg/d) for 2 weeks resulted in increased myocardial Rac1 activity with subsequent hypertension, cardiac hypertrophy, and left ventricular systolic dysfunction in vivo. Isolated right ventricular papillary muscles of T4-treated mice maintained their peak isometric active developed tension but exhibited significant decreases in their corresponding time to peak and in relaxation times. Positive inotropic responses to increasing pacing rate and ß-adrenergic stimulation were also depressed in these muscles. Pravastatin (10 mg/kg/d), a Rac1 inhibitor, significantly decreased myocardial Rac1 activity, hypertension, and cardiomyocyte size in T4-treated mice but could not attenuate gross heart weight or functional cardiac changes in these mice. Our data showed that T4 could activate different signaling pathways with distinct cardiovascular outcomes. We also provide the first mechanistic evidence for the partial involvement of Rac1 activation in T4-induced cardiomyocyte hypertrophy and reveal a putative role for Rac1 in the development of T4-induced hypertension.

Cardiac remodeling caused by transgenic overexpression of a corn Rac gene.

Rac1-GTPase activation plays a key role in the development and progression of cardiac remodeling. Therefore, we engineered a transgenic mouse model by overexpressing cDNA of a constitutively active form of Zea maize Rac gene (ZmRacD) specifically in the hearts of FVB/N mice. Echocardiography and MRI analyses showed cardiac hypertrophy in old transgenic mice, as evidenced by increased left ventricular (LV) mass and LV mass-to-body weight ratio, which are associated with relative ventricular chamber dilation and systolic dysfunction. LV hypertrophy in the hearts of old transgenic mice was further confirmed by an increased heart weight-to-body weight ratio and histopathology analysis. The cardiac remodeling in old transgenic mice was coupled with increased myocardial Rac-GTPase activity (372%) and ROS production (462%). There were also increases in α(1)-integrin (224%) and ß(1)-integrin (240%) expression. This led to the activation of hypertrophic signaling pathways, e.g., ERK1/2 (295%) and JNK (223%). Pravastatin treatment led to inhibition of Rac-GTPase activity and integrin signaling. Interestingly, activation of ZmRacD expression with thyroxin led to cardiac dilation and systolic dysfunction in adult transgenic mice within 2 wk. In conclusion, this is the first study to show the conservation of Rho/Rac proteins between plant and animal kingdoms in vivo. Additionally, ZmRacD is a novel transgenic model that gradually develops a cardiac phenotype with aging. Furthermore, the shift from cardiac hypertrophy to dilated hearts via thyroxin treatment will provide us with an excellent system to study the temporal changes in cardiac signaling from adaptive to maladaptive hypertrophy and heart failure.

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.

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.

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.

Progenitor cell combination normalizes retinal vascular development in the oxygen-induced retinopathy (OIR) model.

Retinopathy of prematurity (ROP) is a disorder of the developing retina of preterm infants. ROP can lead to blindness because of abnormal angiogenesis that is the result of suspended vascular development and vaso-obliteration leading to severe retinal stress and hypoxia. We tested the hypothesis that the use of the human progenitor cell combination, bone marrow-derived CD34+ cells and vascular wall-derived endothelial colony-forming cells (ECFCs), would synergistically protect the developing retinal vasculature in a mouse model of ROP, called oxygen-induced retinopathy (OIR). CD34+ cells alone, ECFCs alone, or the combination thereof were injected intravitreally at either P5 or P12 and pups were euthanized at P17. Retinas from OIR mice injected with ECFCs or the combined treatment revealed formation of the deep vascular plexus (DVP) while still in hyperoxia, with normal-appearing connections between the superficial vascular plexus (SVP) and the DVP. In addition, the combination of cells completely prevented aberrant retinal neovascularization and was more effective anatomically and functionally at rescuing the ischemia phenotype than either cell type alone. We show that the beneficial effects of the cell combination are the result of their ability to orchestrate an acceleration of vascular development and more rapid ensheathment of pericytes on the developing vessels. Lastly, our proteomic and transcriptomic data sets reveal pathways altered by the dual cell therapy, including many involved in neuroretinal maintenance, and principal component analysis (PCA) showed that cell therapy restored OIR retinas to a state that was closely associated with age-matched normal retinas. Together, these data herein support the use of dual cell therapy as a promising preventive treatment for the development of ROP in premature infants.

Enteral Arg-Gln Dipeptide Administration Increases Retinal Docosahexaenoic Acid and Neuroprotectin D1 in a Murine Model of Retinopathy of Prematurity.

Purpose: Low levels of the long chain polyunsaturated fatty acid (LCPUFA) docosahexaenoic acid (DHA) have been implicated in retinopathy of prematurity (ROP). However, oral DHA suffers from poor palatability and is associated with increased bleeding in premature infants. We asked whether oral administration of the neutraceutical arginine-glutamine (Arg-Glu) could increase retinal DHA and improve outcomes in a mouse model of oxygen-induced retinopathy (OIR). Methods: Postnatal day 7 (P7) pups were maintained at 75% oxygen for 5 days and then returned to room air on P12. Pups were gavaged twice daily with Arg-Gln or vehicle from P12 to P17 and eyes were harvested for analysis on P17. Vaso-obliteration and vascular density were assessed on retinal flat mounts and preretinal neovascularization was assessed on retinal cross sections. Retinas were used for measurement of DHA and 10,17S-docosatriene (neuroprotectin D1, NPD1), a key DHA-derived lipid, and for analysis by reverse-phase protein array (RPPA). Results: With Arg-Gln treatment, retinal DHA and NPD1 levels were increased in OIR pups. Arg-Gln reduced preretinal neovascularization by 39 ± 6% (P < 0.05) relative to vehicle control. This was accompanied by a restoration of vascular density of the retina in the pups treated with Arg-Gln (73.0 ± 3.0%) compared to vehicle (53.1 ± 3.4%; P < 0.05). Arg-Gln dipeptide restored OIR-induced signaling changes toward normoxia and was associated with normalization of insulin-like growth factor receptor 1 signaling and reduction of apoptosis and an increase in anti-apoptosis proteins. Conclusions: Arg-Gln may serve as a safer and easily tolerated nutraceutical agent for prevention or treatment of ROP.

Restructuring of the Gut Microbiome by Intermittent Fasting Prevents Retinopathy and Prolongs Survival in db/db Mice.

Intermittent fasting (IF) protects against the development of metabolic diseases and cancer, but whether it can prevent diabetic microvascular complications is not known. In db/db mice, we examined the impact of long-term IF on diabetic retinopathy (DR). Despite no change in glycated hemoglobin, db/db mice on the IF regimen displayed significantly longer survival and a reduction in DR end points, including acellular capillaries and leukocyte infiltration. We hypothesized that IF-mediated changes in the gut microbiota would produce beneficial metabolites and prevent the development of DR. Microbiome analysis revealed increased levels of Firmicutes and decreased Bacteroidetes and Verrucomicrobia. Compared with db/db mice on ad libitum feeding, changes in the microbiome of the db/db mice on IF were associated with increases in gut mucin, goblet cell number, villi length, and reductions in plasma peptidoglycan. Consistent with the known modulatory effects of Firmicutes on bile acid (BA) metabolism, measurement of BAs demonstrated a significant increase of tauroursodeoxycholate (TUDCA), a neuroprotective BA, in db/db on IF but not in db/db on AL feeding. TGR5, the TUDCA receptor, was found in the retinal primary ganglion cells. Expression of TGR5 did not change with IF or diabetes. However, IF reduced retinal TNF-α mRNA, which is a downstream target of TGR5 activation. Pharmacological activation of TGR5 using INT-767 prevented DR in a second diabetic mouse model. These findings support the concept that IF prevents DR by restructuring the microbiota toward species producing TUDCA and subsequent retinal protection by TGR5 activation.

Reactive oxygen species in vascular endothelial cell motility. Roles of NAD(P)H oxidase and Rac1.

Reactive oxygen species (ROS) are acknowledged generally to be multi-faceted regulators of cellular functions that trigger various pathological states when present chronically or transiently at non-physiologically high levels. Here we focus on the physiological involvement of ROS in cellular motility, with special emphasis on endothelial cells (EC). An important source of ROS within EC is the non-phagocytic NAD(P)H oxidase, and the small GTPase Rac1 plays a central role in activating this complex. Rac1 is one of the three Rho-family molecules (Rac, Rho and Cdc42) involved in the control of the actin cytoskeleton in response to various signals. In this review we examine the evidence linking ROS production, Rac1 activation and actin organization to EC motility, considering mechanisms for direct interaction of ROS and actin and the effects of ROS on proteins that regulate the actin cytoskeleton. The accumulated evidence suggests that ROS are important regulators of the actin cytoskeletal dynamics and cellular motility, and more in-depth studies are needed to understand the underlying mechanisms.

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.

Bone Marrow-Derived Cell Recruitment to the Neurosensory Retina and Retinal Pigment Epithelial Cell Layer Following Subthreshold Retinal Phototherapy.

Purpose: We investigated whether subthreshold retinal phototherapy (SRPT) was associated with recruitment of bone marrow (BM)-derived cells to the neurosensory retina (NSR) and RPE layer. Methods: GFP chimeric mice and wild-type (WT) mice were subjected to SRPT using a slit-lamp infrared laser. Duty cycles of 5%, 10%, 15%, and 20% (0.1 seconds, 250 mW, spot size 50 µm) with 30 applications were placed 50 to 100 µm from the optic disc. In adoptive transfer studies, GFP+ cells were given intravenously immediately after WT mice received SRPT. Immunohistochemistry was done for ionized calcium-binding adapter molecule-1 (IBA-1+), CD45, Griffonia simplicifolia lectin isolectin B4, GFP or cytokeratin). Expression of Ccl2, Il1b, Il6, Hspa1a, Hsp90aa1, Cryab, Hif1a, Cxcl12, and Cxcr4 mRNA and flow cytometry of the NSR and RPE-choroid were performed. Results: Within 12 to 24 hours of SRPT, monocytes were detected in the NSR and RPE-choroid. Detection of reparative progenitors in the RPE occurred at 2 weeks using flow cytometry. Recruitment of GFP+ cells to the RPE layer occurred in a duty cycle-dependent manner in chimeric mice and in mice undergoing adoptive transfer. Hspa1a, Hsp90aa1, and Cryab mRNAs increased in the NSR at 2 hours post laser; Hif1a, Cxcl12, Hspa1a increased at 4 hours in the RPE-choroid; and Ccl2, Il1b, Ifng, and Il6 increased at 12 to 24 hours in the RPE-choroid. Conclusions: SRPT induces monocyte recruitment to the RPE followed by hematopoietic progenitor cell homing at 2 weeks. Recruitment occurs in a duty cycle-dependent manner and potentially could contribute to the therapeutic efficacy of SRPT.