Tibetan PHD2, an allele with loss-of-function properties.

Tibetans have adapted to the chronic hypoxia of high altitude and display a distinctive suite of physiologic adaptations, including augmented hypoxic ventilatory response and resistance to pulmonary hypertension. Genome-wide studies have consistently identified compelling genetic signatures of natural selection in two genes of the Hypoxia Inducible Factor pathway, PHD2 and HIF2A The product of the former induces the degradation of the product of the latter. Key issues regarding Tibetan PHD2 are whether it is a gain-of-function or loss-of-function allele, and how it might contribute to high-altitude adaptation. Tibetan PHD2 possesses two amino acid changes, D4E and C127S. We previously showed that in vitro, Tibetan PHD2 is defective in its interaction with p23, a cochaperone of the HSP90 pathway, and we proposed that Tibetan PHD2 is a loss-of-function allele. Here, we report that additional PHD2 mutations at or near Asp-4 or Cys-127 impair interaction with p23 in vitro. We find that mice with the Tibetan Phd2 allele display augmented hypoxic ventilatory response, supporting this loss-of-function proposal. This is phenocopied by mice with a mutation in p23 that abrogates the PHD2:p23 interaction. Hif2a haploinsufficiency, but not the Tibetan Phd2 allele, ameliorates hypoxia-induced increases in right ventricular systolic pressure. The Tibetan Phd2 allele is not associated with hemoglobin levels in mice. We propose that Tibetans possess genetic alterations that both activate and inhibit selective outputs of the HIF pathway to facilitate successful adaptation to the chronic hypoxia of high altitude.

No evidence of Gremlin1-mediated activation of VEGFR2 signaling in endothelial cells.

Canonical Gremlin1 (GREM1) signaling involves binding to and sequestering bone morphogenetic proteins (BMPs) in the extracellular matrix, preventing the activation of cognate BMP receptor. Exquisite temporospatial control of the GREM1-BMP interaction is required during development, and perturbation of this balance leads to abnormal limb formation and defective kidney development. In addition to inhibition of BMP signaling, several other noncanonical signaling modalities of GREM1 have been postulated. Some literature reports have suggested that GREM1 can bind to and activate vascular endothelial growth factor receptor-2 (VEGFR2) in endothelial cells, human kidney epithelial cells, and others. These reports suggest that the GREM1 → VEGFR2 signaling can drive angiogenesis both in vitro and in vivo We report here that, despite exhaustive attempts, we did not observe GREM1 activation of VEGFR2 in any of the cell lines reported by the above-mentioned studies. Incubation of endothelial colony-forming cells (ECFCs) or human umbilical vein endothelial cells (HUVECs) with recombinant VEGF triggered a robust increase in VEGFR2 tyrosine phosphorylation. In contrast, no VEGFR2 phosphorylation was detected when cells were incubated with recombinant GREM1 over a range of time points and concentrations. We also show that GREM1 does not interfere with VEGF-mediated VEGFR2 activation, suggesting that GREM1 does not bind with any great affinity to VEGFR2. Measurements of ECFC barrier integrity revealed that VEGF induces barrier function disruption, but recombinant human GREM1 had no effect in this assay. We believe that these results provide an important clarification of the potential interaction between GREM1 and VEGFR2 in mammalian cells.

The Vasoreparative Function of Myeloid Angiogenic Cells Is Impaired in Diabetes Through the Induction of IL1ß.

Myeloid angiogenic cells (MACs) promote revascularization through the paracrine release of angiogenic factors and have been harnessed as therapeutic cells for many ischemic diseases. However, their proangiogenic properties have been suggested to be diminished in diabetes. This study investigates how the diabetic milieu affects the immunophenotype and function of MACs. Both MACs isolated from diabetic conditions and healthy cells exposed to a diabetic environment were used to determine the potential of MACs as a cell therapy for diabetic-related ischemia. MACs were isolated from human peripheral blood and characterized alongside proinflammatory macrophages M (LPS + IFNγ) and proangiogenic macrophages M (IL4). Functional changes in MACs in response to high-d-glucose were assessed using an in vitro 3D-tubulogenesis assay. Phenotypic changes were determined by gene and protein expression analysis. Additionally, MACs from type 1 diabetic (T1D) patients and corresponding controls were isolated and characterized. Our evidence demonstrates MACs identity as a distinct macrophage subtype that shares M2 proangiogenic characteristics, but can be distinguished by CD163hi expression. High-d-glucose treatment significantly reduced MACs proangiogenic capacity, which was associated with a significant increase in IL1ß mRNA and protein expression. Inhibition of IL1ß abrogated the antiangiogenic effect induced by high-d-glucose. IL1ß was also significantly upregulated in MACs isolated from T1D patients with microvascular complications compared to T1D patients without microvascular complications or nondiabetic volunteers. This study demonstrates that Type 1 diabetes and diabetic-like conditions impair the proangiogenic and regenerative capacity of MACs, and this response is mediated by IL-1ß. Stem Cells 2018;36:834-843.

The vasoreparative potential of endothelial colony-forming cells in the ischemic retina is enhanced by cibinetide, a non-hematopoietic erythropoietin mimetic.

PURPOSE: Retinal ischemia remains a common sight threatening end-point in blinding diseases such as diabetic retinopathy and retinopathy of prematurity. Endothelial colony forming cells (ECFCs) represent a subpopulation of endothelial progenitors with therapeutic utility for promoting reparative angiogenesis in the ischaemic retina. The current study has investigated the potential of enhancing this cell therapy approach by the dampening of the pro-inflammatory milieu typical of ischemic retina. Based on recent findings that ARA290 (cibinetide), a peptide based on the Helix-B domain of erythropoietin (EPO), is anti-inflammatory and tissue-protective, the effect of this peptide on ECFC-mediated vascular regeneration was studied in the ischemic retina. METHODS: The effects of ARA290 on pro-survival signaling and function were assessed in ECFC cultures in vitro. Efficacy of ECFC transplantation therapy to promote retinal vascular repair in the presence and absence of ARA290 was studied in the oxygen induced retinopathy (OIR) model of retinal ischemia. The inflammatory cytokine profile and microglial activation were studied as readouts of inflammation. RESULTS: ARA290 activated pro-survival signaling and enhanced cell viability in response to H2O2-mediated oxidative stress in ECFCs in vitro. Preconditioning of ECFCs with EPO or ARA290 prior to delivery to the ischemic retina did not enhance vasoreparative function. ARA290 delivered systemically to OIR mice reduced pro-inflammatory expression of IL-1ß and TNF-α in the mouse retina. Following intravitreal transplantation, ECFCs incorporated into the damaged retinal vasculature and significantly reduced avascular area. The vasoreparative function of ECFCs was enhanced in the presence of ARA290 but not EPO. DISCUSSION: Regulation of the pro-inflammatory milieu of the ischemic retina can be enhanced by ARA290 and may be a useful adjunct to ECFC-based cell therapy for ischemic retinopathies.

MicroRNA-containing extracellular vesicles released from endothelial colony-forming cells modulate angiogenesis during ischaemic retinopathy.

Endothelial colony-forming cells (ECFCs) are a defined subtype of endothelial progenitors that modulate vascular repair and promote perfusion in ischaemic tissues. Their paracrine activity on resident vasculature is ill-defined, but mediated, at least in part, by the transfer of extracellular vesicles (EVs). To evaluate the potential of isolated EVs to provide an alternative to cell-based therapies, we first performed a physical and molecular characterization of those released by ECFCs. Their effects upon endothelial cells in vitro and angiogenesis in vivo in a model of proliferative retinopathy were assessed. The EVs expressed typical markers CD9 and CD63 and formed a heterogeneous population ranging in size from ~60 to 1500 nm by electron microscopy. ECFC EVs were taken up by endothelial cells and increased cell migration. This was reflected by microarray analyses which showed significant changes in expression of genes associated with angiogenesis. Sequencing of small RNAs in ECFCs and their EVs showed that multiple microRNAs are highly expressed and concentrated in EVs. The functional categories significantly enriched for the predicted target genes of these microRNAs included angiogenesis. Intravitreally delivered ECFC EVs were associated with the vasculature and significantly reduced the avascular area in a mouse oxygen-induced retinopathy model. Our findings confirm the potential of isolated EVs to influence endothelial cell function and act as a therapy to modulate angiogenesis. The functions associated with the specific microRNAs detected in ECFC EVs support a role for microRNA transfer in mediating the observed effects.

Ex vivo expansion of human outgrowth endothelial cells leads to IL-8-mediated replicative senescence and impaired vasoreparative function.

Harnessing outgrowth endothelial cells (OECs) for vasoreparative therapy and tissue engineering requires efficient ex vivo expansion. How such expansion impacts on OEC function is largely unknown. In this study, we show that OECs become permanently cell-cycle arrested after ex vivo expansion, which is associated with enlarged cell size, ß-galactosidase activity, DNA damage, tumor suppressor pathway activation, and significant transcriptome changes. These senescence hallmarks were coupled with low telomerase activity and telomere shortening, indicating replicative senescence. OEC senescence limited their regenerative potential by impairing vasoreparative properties in vitro and in vivo. Integrated transcriptome-proteome analysis identified inflammatory signaling pathways as major mechanistic components of the OEC senescence program. In particular, IL8 was an important facilitator of this senescence; depletion of IL8 in OECs significantly extended ex vivo lifespan, delayed replicative senescence, and enhanced function. While the ability to expand OEC numbers prior to autologous or allogeneic therapy remains a useful property, their replicative senescence and associated impairment of vasorepair needs to be considered. This study also suggests that modulation of the senescence-associated secretory phenotype could be used to optimize OEC therapy.

Vasculogenic and osteogenesis-enhancing potential of human umbilical cord blood endothelial colony-forming cells.

Umbilical cord blood-derived endothelial colony-forming cells (UCB-ECFC) show utility in neovascularization, but their contribution to osteogenesis has not been defined. Cocultures of UCB-ECFC with human fetal-mesenchymal stem cells (hfMSC) resulted in earlier induction of alkaline phosphatase (ALP) (Day 7 vs. 10) and increased mineralization (1.9×; p < .001) compared to hfMSC monocultures. This effect was mediated through soluble factors in ECFC-conditioned media, leading to 1.8-2.2× higher ALP levels and a 1.4-1.5× increase in calcium deposition (p < .01) in a dose-dependent manner. Transcriptomic and protein array studies demonstrated high basal levels of osteogenic (BMPs and TGF-ßs) and angiogenic (VEGF and angiopoietins) regulators. Comparison of defined UCB and adult peripheral blood ECFC showed higher osteogenic and angiogenic gene expression in UCB-ECFC. Subcutaneous implantation of UCB-ECFC with hfMSC in immunodeficient mice resulted in the formation of chimeric human vessels, with a 2.2-fold increase in host neovascularization compared to hfMSC-only implants (p = .001). We conclude that this study shows that UCB-ECFC have potential in therapeutic angiogenesis and osteogenic applications in conjunction with MSC. We speculate that UCB-ECFC play an important role in skeletal and vascular development during perinatal development but less so in later life when expression of key osteogenesis and angiogenesis genes in ECFC is lower.

Advances in our understanding of diabetic retinopathy.

Diabetic retinopathy remains the most common complication of diabetes mellitus and is a leading cause of visual loss in industrialized nations. The clinicopathology of the diabetic retina has been extensively studied, although the precise pathogenesis and cellular and molecular defects that lead to retinal vascular, neural and glial cell dysfunction remain somewhat elusive. This lack of understanding has seriously limited the therapeutic options available for the ophthalmologist and there is a need to identify the definitive pathways that initiate retinal cell damage and drive progression to overt retinopathy. The present review begins by outlining the natural history of diabetic retinopathy, the clinical features and risk factors. Reviewing the histopathological data from clinical specimens and animal models, the recent paradigm that neuroretinal dysfunction may play an important role in the early development of the disease is discussed. The review then focuses on the molecular pathogenesis of diabetic retinopathy with perspective provided on new advances that have furthered our understanding of the key mechanisms underlying early changes in the diabetic retina. Studies have also emerged in the past year suggesting that defective repair of injured retinal vessels by endothelial progenitor cells may contribute to the pathogenesis of diabetic retinopathy. We assess these findings and discuss how they could eventually lead to new therapeutic options for diabetic retinopathy.

Human blood vessel organoids reveal a critical role for CTGF in maintaining microvascular integrity.

The microvasculature plays a key role in tissue perfusion and exchange of gases and metabolites. In this study we use human blood vessel organoids (BVOs) as a model of the microvasculature. BVOs fully recapitulate key features of the human microvasculature, including the reliance of mature endothelial cells on glycolytic metabolism, as concluded from metabolic flux assays and mass spectrometry-based metabolomics using stable tracing of 13C-glucose. Pharmacological targeting of PFKFB3, an activator of glycolysis, using two chemical inhibitors results in rapid BVO restructuring, vessel regression with reduced pericyte coverage. PFKFB3 mutant BVOs also display similar structural remodelling. Proteomic analysis of the BVO secretome reveal remodelling of the extracellular matrix and differential expression of paracrine mediators such as CTGF. Treatment with recombinant CTGF recovers microvessel structure. In this work we demonstrate that BVOs rapidly undergo restructuring in response to metabolic changes and identify CTGF as a critical paracrine regulator of microvascular integrity.

PLAC8-Mediated Activation of NOX4 Signalling Restores Angiogenic Function of Endothelial Colony-Forming Cells in Experimental Hypoxia.

Ischaemic cardiovascular disease is associated with tissue hypoxia as a significant determinant of angiogenic dysfunction and adverse remodelling. While cord blood-derived endothelial colony-forming cells (CB-ECFCs) hold clear therapeutic potential due to their enhanced angiogenic and proliferative capacity, their impaired functionality within the disease microenvironment represents a major barrier to clinical translation. The aim of this study was to define the specific contribution of NOX4 NADPH oxidase, which we previously reported as a key CB-ECFC regulator, to hypoxia-induced dysfunction and its potential as a therapeutic target. CB-ECFCs exposed to experimental hypoxia demonstrated downregulation of NOX4-mediated reactive oxygen species (ROS) signalling linked with a reduced tube formation, which was partially restored by NOX4 plasmid overexpression. siRNA knockdown of placenta-specific 8 (PLAC8), identified by microarray analysis as an upstream regulator of NOX4 in hypoxic versus normoxic CB-ECFCs, enhanced tube formation, NOX4 expression and hydrogen peroxide generation, and induced several key transcription factors associated with downstream Nrf2 signalling. Taken together, these findings indicated that activation of the PLAC8-NOX4 signalling axis improved CB-ECFC angiogenic functions in experimental hypoxia, highlighting this pathway as a potential target for protecting therapeutic cells against the ischaemic cardiovascular disease microenvironment.

Selective extracellular vesicle-mediated export of an overlapping set of microRNAs from multiple cell types.

BACKGROUND: MicroRNAs (miRNAs) are a class of small RNA molecules that regulate expression of specific mRNA targets. They can be released from cells, often encapsulated within extracellular vesicles (EVs), and therefore have the potential to mediate intercellular communication. It has been suggested that certain miRNAs may be selectively exported, although the mechanism has yet to be identified. Manipulation of the miRNA content of EVs will be important for future therapeutic applications. We therefore wished to assess which endogenous miRNAs are enriched in EVs and how effectively an overexpressed miRNA would be exported. RESULTS: Small RNA libraries from HEK293T cells and vesicles before or after transfection with a vector for miR-146a overexpression were analysed by deep sequencing. A subset of miRNAs was found to be enriched in EVs; pathway analysis of their predicted target genes suggests a potential role in regulation of endocytosis. RT-qPCR in additional cell types and analysis of publicly available data revealed that many of these miRNAs tend to be widely preferentially exported. Whilst overexpressed miR-146a was highly enriched both in transfected cells and their EVs, the cellular:EV ratios of endogenous miRNAs were not grossly altered. MiR-451 was consistently the most highly exported miRNA in many different cell types. Intriguingly, Argonaute2 (Ago2) is required for miR-451 maturation and knock out of Ago2 has been shown to decrease expression of other preferentially exported miRNAs (eg miR-150 and miR-142-3p). CONCLUSION: The global expression data provided by deep sequencing confirms that specific miRNAs are enriched in EVs released by HEK293T cells. Observation of similar patterns in a range of cell types suggests that a common mechanism for selective miRNA export may exist.

Deep sequencing reveals predominant expression of miR-21 amongst the small non-coding RNAs in retinal microvascular endothelial cells.

The retinal vascular endothelium is essential for angiogenesis and is involved in maintaining barrier selectivity and vascular tone. The aim of this study was to identify and quantify microRNAs and other small regulatory non-coding RNAs (ncRNAs) which may regulate these crucial functions. Primary bovine retinal microvascular endothelial cells (RMECs) provide a well-characterized in vitro system for studying angiogenesis. RNA extracted from RMECs was used to prepare a small RNA library for deep sequencing (Illumina Genome Analyzer). A total of 6.8 million reads were mapped to 250 known microRNAs in miRBase (release 16). In many cases, the most frequent isomiR differed from the sequence reported in miRBase. In addition, five novel microRNAs, 13 novel bovine orthologs of known human microRNAs and multiple new members of the miR-2284/2285 family were detected. Several ∼30 nucleotide sno-miRNAs were identified, with the most highly expressed being derived from snoRNA U78. Highly expressed microRNAs previously associated with endothelial cells included miR-126 and miR-378, but the most highly expressed was miR-21, comprising more than one-third of all mapped reads. Inhibition of miR-21 with an LNA inhibitor significantly reduced proliferation, migration, and tube-forming capacity of RMECs. The independence from prior sequence knowledge provided by deep sequencing facilitates analysis of novel microRNAs and other small RNAs. This approach also enables quantitative evaluation of microRNA expression, which has highlighted the predominance of a small number of microRNAs in RMECs. Knockdown of miR-21 suggests a role for this microRNA in regulation of angiogenesis in the retinal microvasculature.

Advances in cell therapies using stem cells/progenitors as a novel approach for neurovascular repair of the diabetic retina.

BACKGROUND: Diabetic retinopathy, a major complication of diabetes mellitus, is a leading cause of sigh-loss in working age adults. Progressive loss of integrity of the retinal neurovascular unit is a central element in the disease pathogenesis. Retinal ischemia and inflammatory processes drive interrelated pathologies such as blood retinal barrier disruption, fluid accumulation, gliosis, neuronal loss and/or aberrant neovascularisation. Current treatment options are somewhat limited to late-stages of the disease where there is already significant damage to the retinal architecture arising from degenerative, edematous and proliferative pathology. New preventive and interventional treatments to target early vasodegenerative and neurodegenerative stages of the disease are needed to ensure avoidance of sight-loss. MAIN BODY: Historically, diabetic retinopathy has been considered a primarily microvascular disease of the retina and clinically it is classified based on the presence and severity of vascular lesions. It is now known that neurodegeneration plays a significant role during the pathogenesis. Loss of neurons has been documented at early stages in pre-clinical models as well as in individuals with diabetes and, in some, even prior to the onset of clinically overt diabetic retinopathy. Recent studies suggest that some patients have a primarily neurodegenerative phenotype. Retinal pigment epithelial cells and the choroid are also affected during the disease pathogenesis and these tissues may also need to be addressed by new regenerative treatments. Most stem cell research for diabetic retinopathy to date has focused on addressing vasculopathy. Pre-clinical and clinical studies aiming to restore damaged vasculature using vasoactive progenitors including mesenchymal stromal/stem cells, adipose stem cells, CD34+ cells, endothelial colony forming cells and induced pluripotent stem cell derived endothelial cells are discussed in this review. Stem cells that could replace dying neurons such as retinal progenitor cells, pluripotent stem cell derived photoreceptors and ganglion cells as well as Müller stem cells are also discussed. Finally, challenges of stem cell therapies relevant to diabetic retinopathy are considered. CONCLUSION: Stem cell therapies hold great potential to replace dying cells during early and even late stages of diabetic retinopathy. However, due to the presence of different phenotypes, selecting the most suitable stem cell product for individual patients will be crucial for successful treatment.

Long term high glucose exposure induces premature senescence in retinal endothelial cells.

Purpose: Features of cellular senescence have been described in diabetic retinal vasculature. The aim of this study was to investigate how the high glucose microenvironment impacts on the senescence program of retinal endothelial cells. Methods: Human retinal microvascular endothelial cells were cultured under control and high glucose conditions of 5 mM and 25 mM D-glucose, respectively. Isomeric l-glucose was used as the osmotic control. Cells were counted using CASY technology until they reached their Hayflick limit. Senescence-associated ß-Galactosidase was used to identify senescent cells. Endothelial cell functionality was evaluated by the clonogenic, 3D tube formation, and barrier formation assays. Cell metabolism was characterized using the Seahorse Bioanalyzer. Gene expression analysis was performed by bulk RNA sequencing. Retinal tissues from db/db and db/+ mice were evaluated for the presence of senescent cells. Publicly available scRNA-sequencing data for retinas from Akimba and control mice was used for gene set enrichment analysis. Results: Long term exposure to 25 mM D-Glucose accelerated the establishment of cellular senescence in human retinal endothelial cells when compared to 5 mM D-glucose and osmotic controls. This was shown from 4 weeks, by a significant slower growth, higher percentages of cells positive for senescence-associated ß-galactosidase, an increase in cell size, and lower expression of pRb and HMGB2. These senescence features were associated with decreased clonogenic capacity, diminished tubulogenicity, and impaired barrier function. Long term high glucose-cultured cells exhibited diminished glycolysis, with lower protein expression of GLUT1, GLUT3, and PFKFB3. Transcriptomic analysis, after 4 weeks of culture, identified downregulation of ALDOC, PFKL, and TPI1, in cells cultured with 25 mM D-glucose when compared to controls. The retina from db/db mice showed a significant increase in acellular capillaries associated with a significant decrease in vascular density in the intermediate and deep retinal plexuses, when compared to db/+ mice. Senescent endothelial cells within the db/db retinal vasculature were identified by senescence-associated ß-galactosidase staining. Analysis of single cell transcriptomics data for the Akimba mouse retina highlighted an enrichment of senescence and senescence-associated secretory phenotype gene signatures when compared to control mice. Conclusion: A diabetic-like microenvironment of 25 mM D-glucose was sufficient to accelerate the establishment of cellular senescence in human retinal microvascular endothelial cells.

Retinal pigment epithelium extracellular vesicles are potent inducers of age-related macular degeneration disease phenotype in the outer retina.

Age-related macular degeneration (AMD) is a leading cause of blindness. Vision loss is caused by the retinal pigment epithelium (RPE) and photoreceptors atrophy and/or retinal and choroidal angiogenesis. Here we use AMD patient-specific RPE cells with the Complement Factor H Y402H high-risk polymorphism to perform a comprehensive analysis of extracellular vesicles (EVs), their cargo and role in disease pathology. We show that AMD RPE is characterised by enhanced polarised EV secretion. Multi-omics analyses demonstrate that AMD RPE EVs carry RNA, proteins and lipids, which mediate key AMD features including oxidative stress, cytoskeletal dysfunction, angiogenesis and drusen accumulation. Moreover, AMD RPE EVs induce amyloid fibril formation, revealing their role in drusen formation. We demonstrate that exposure of control RPE to AMD RPE apical EVs leads to the acquisition of AMD features such as stress vacuoles, cytoskeletal destabilization and abnormalities in the morphology of the nucleus. Retinal organoid treatment with apical AMD RPE EVs leads to disrupted neuroepithelium and the appearance of cytoprotective alpha B crystallin immunopositive cells, with some co-expressing retinal progenitor cell markers Pax6/Vsx2, suggesting injury-induced regenerative pathways activation. These findings indicate that AMD RPE EVs are potent inducers of AMD phenotype in the neighbouring RPE and retinal cells.

Myeloid angiogenic cells act as alternative M2 macrophages and modulate angiogenesis through interleukin-8.

Endothelial progenitor cells (EPCs) promote angiogenesis, and clinical trials have shown such cell therapy to be feasible for treating ischemic disease. However, clinical outcomes have been contradictory owing to the diverse range of EPC types used. We recently characterized two EPC subtypes, and identified outgrowth endothelial cells as the only EPC type with true progenitor and endothelial characteristics. By contrast, myeloid angiogenic cells (MACs) were shown to be monocytic cells without endothelial characteristics despite being widely described as "EPCs." In the current study we demonstrated that although MACs do not become endothelial cells or directly incorporate into a microvascular network, they can significantly induce endothelial tube formation in vitro and vascular repair in vivo. MAC-derived interleukin-8 (IL-8) was identified as a key paracrine factor, and blockade of IL-8 but not vascular endothelial growth factor (VEGF) prevented MAC-induced angiogenesis. Extracellular IL-8 transactivates VEGFR2 and induces phosphorylation of extracellular signal-regulated kinases. Further transcriptomic and immunophenotypic analysis indicates that MACs represent alternative activated M2 macrophages. Our findings demonstrate an unequivocal role for MACs in angiogenesis, which is linked to paracrine release of cytokines such as IL-8. We also show, for the first time, the true identity of these cells as alternative M2 macrophages with proangiogenic, antiinflammatory and pro-tissue-repair properties.

The Long Pentraxin PTX3 as a New Biomarker and Pharmacological Target in Age-Related Macular Degeneration and Diabetic Retinopathy.

Age related macular degeneration (AMD) and diabetic retinopathy (DR) are multifactorial, neurodegenerative and inflammatory diseases of the eye primarily involving cellular and molecular components of the outer and inner blood-retina barriers (BRB), respectively. Largely contributed by genetic factors, particularly polymorphisms in complement genes, AMD is a paradigm of retinal immune dysregulation. DR, a major complication of diabetes mellitus, typically presents with increased vascular permeability and occlusion of the retinal vasculature that leads, in the proliferative form of the disease, to neovascularization, a pathogenic trait shared with advanced AMD. In spite of distinct etiology and clinical manifestations, both pathologies share common drivers, such as chronic inflammation, either of immune (in AMD) or metabolic (in DR) origin, which initiates and propagates degeneration of the neural retina, yet the underlying mechanisms are still unclear. As a soluble pattern recognition molecule with complement regulatory functions and a marker of vascular damage, long pentraxin 3 (PTX3) is emerging as a novel player in ocular homeostasis and a potential pharmacological target in neurodegenerative disorders of the retina. Physiologically present in the human eye and induced in inflammatory conditions, this protein is strategically positioned at the BRB interface, where it acts as a "molecular trap" for complement, and modulates inflammation both in homeostatic and pathological conditions. Here, we discuss current viewpoints on PTX3 and retinal diseases, with a focus on AMD and DR, the roles therein proposed for this pentraxin, and their implications for the development of new therapeutic strategies.

Current concepts on endothelial stem cells definition, location, and markers.

Ischemic vascular disease is a major cause of mortality and morbidity worldwide, and regeneration of blood vessels in perfusion-deficient tissues is a worthwhile therapeutic goal. The idea of delivering endothelial stem/progenitor cells to repair damaged vasculature, reperfuse hypoxic tissue, prevent cell death, and consequently diminish tissue inflammation and fibrosis has a strong scientific basis and clinical value. Various labs have proposed endothelial stem/progenitor cell candidates. This has created confusion, as there are profound differences between these cell definitions based on isolation methodology, characterization, and reparative biology. Here, a stricter definition based on stem cell biology principles is proposed. Although preclinical studies have often been promising, results from clinical trials have been highly contradictory and served to highlight multiple challenges associated with disappointing therapeutic benefit. This article reviews recent accomplishments in the field and discusses current difficulties when developing endothelial stem cell therapies. Emerging evidence that disputes the classic view of the bone marrow as the source for these cells and supports the vascular wall as the niche for these tissue-resident endothelial stem cells is considered. In addition, novel markers to identify endothelial stem cells, including CD157, EPCR, and CD31low VEGFR2low IL33+ Sox9+ , are described.

miR-130a activates the VEGFR2/STAT3/HIF1α axis to potentiate the vasoregenerative capacity of endothelial colony-forming cells in hypoxia.

Hypoxia modulates reparative angiogenesis, which is a tightly regulated pathophysiological process. MicroRNAs (miRNAs) are important regulators of gene expression in hypoxia and angiogenesis. However, we do not yet have a clear understanding of how hypoxia-induced miRNAs fine-tune vasoreparative processes. Here, we identify miR-130a as a mediator of the hypoxic response in human primary endothelial colony-forming cells (ECFCs), a well-characterized subtype of endothelial progenitors. Under hypoxic conditions of 1% O2, miR-130a gain-of-function enhances ECFC pro-angiogenic capacity in vitro and potentiates their vasoreparative properties in vivo. Mechanistically, miR-130a orchestrates upregulation of VEGFR2, activation of STAT3, and accumulation of HIF1α via translational inhibition of Ddx6. These findings unveil a new role for miR-130a in hypoxia, whereby it activates the VEGFR2/STAT3/HIF1α axis to enhance the vasoregenerative capacity of ECFCs.

Transcriptional regulation of a brown adipocyte-specific gene, UCP1, by KLF11 and KLF15.

Several growth factors and transcription factors have been reported to play important roles in brown adipocyte differentiation and modulation of thermogenic gene expression, especially the expression of UCP1. In this study, we focused on KLF11 and KLF15, which were expressed highly in brown adipose tissue. Our data demonstrated that KLF11 and KLF15 interacted directly with the UCP1 promoter using GC-box and GT-boxes, respectively. Co-transfection of KLF11 and KLF15 in the mesenchymal stem cell line muBM3.1 during brown adipocyte differentiation enhanced the expression level of UCP1. KLF11, but not KLF15, was essential for UCP1 expression during brown adipocyte differentiation of muBM3.1.