Rational Transplantation of human CD34+ stem cells to ischemic tissues has been associated with reduced angina, improved exercise time and reduced amputation rates in phase 2 clinical trials and has been shown to induce neo-vascularization in pre-clinical models. Previous studies have suggested that paracrine factors secreted by these pro-angiogenic cells are responsible, at least in part, for the angiogenic effects induced by CD34+ cell transplantation. Objective Our objective was to investigate the mechanism of CD34+ stem cell induced pro-angiogenic paracrine effects and to examine if exosomes, a component of paracrine secretion, are involved. Methods and Results Exosomes collected from the conditioned media of mobilized human CD34+ cells had the characteristic size (40–90 nm; determined via dynamic light scattering), cup-shaped morphology (electron microscopy), expressed exosome-marker proteins CD63, phosphatidylserine (flow cytometry) and TSG101 (immunoblotting), besides expressing CD34+ cell lineage marker protein, CD34. In vitro, CD34+ exosomes replicated the angiogenic activity of CD34+ cells by increasing endothelial cell viability, proliferation and tube formation on Matrigel. In vivo, the CD34+ exosomes stimulated angiogenesis in Matrigel plug and corneal assays. Interestingly, exosomes from CD34+ cells, but not from CD34+ cell-depleted mononuclear cells had angiogenic activity. Conclusions Our data demonstrate that human CD34+ cells secrete exosomes that have independent angiogenic activity both in vitro and in vivo. CD34+ exosomes may represent a significant component of the paracrine effect of progenitor-cell transplantation for therapeutic angiogenesis.

Rationale Ischemic cardiovascular disease represents one of the largest epidemics currently facing the aging population. Current literature has illustrated the efficacy of autologous, stem cell therapies as novel strategies for treating these disorders. The CD34+ hematopoetic stem cell has shown significant promise in addressing myocardial ischemia by promoting angiogenesis that helps preserve the functionality of ischemic myocardium. Unfortunately, both viability and angiogenic quality of autologous CD34+ cells decline with advanced age and diminished cardiovascular health. Objective To offset age and health-related angiogenic declines in CD34+ cells, we explored whether the therapeutic efficacy of human CD34+ cells could be enhanced by augmenting their secretion of the known angiogenic factor, sonic hedgehog (Shh). Methods and Results When injected into the border zone of mice following acute myocardial infarction (AMI), Shh-modified CD34+ cells (CD34Shh) protected against ventricular dilation and cardiac functional declines associated with AMI. Treatment with CD34Shh also reduced infarct size and increased border zone capillary density compared to unmodified CD34 cells or cells transfected with the empty vector. CD34Shh primarily store and secrete Shh protein in exosomes and this storage process appears to be cell-type specific. In vitro analysis of exosomes derived from CD34Shh revealed that; 1) exosomes transfer Shh protein to other cell types and, 2) exosomal transfer of functional Shh elicits induction of the canonical Shh signaling pathway in recipient cells. Conclusions Exosome-mediated delivery of Shh to ischemic myocardium represents a major mechanism explaining the observed preservation of cardiac function in mice treated with CD34Shh cells.

Polymyxins are antibiotics used in the last line of defense to combat multidrug-resistant infections by Gram-negative bacteria. Polymyxin resistance arises through charge modification of the bacterial outer membrane with the attachment of the cationic sugar 4-amino-4-deoxy-L-arabinose to lipid A, a reaction catalyzed by the integral membrane lipid-to-lipid glycosyltransferase 4-amino-4-deoxy-L-arabinose transferase (ArnT). Here, we report crystal structures of ArnT from Cupriavidus metallidurans, alone and in complex with the lipid carrier undecaprenyl phosphate, at 2.8 and 3.2 angstrom resolution, respectively. The structures show cavities for both lipidic substrates, which converge at the active site. A structural rearrangement occurs on undecaprenyl phosphate binding, which stabilizes the active site and likely allows lipid A binding. Functional mutagenesis experiments based on these structures suggest a mechanistic model for ArnT family enzymes.

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Normal vision requires the precise control of vascular growth to maintain corneal transparency. Here we provide evidence for a unique mechanism by which the Forkhead box transcription factor FoxC1 regulates corneal vascular development. Murine Foxc1 is essential for development of the ocular anterior segment, and in humans, mutations have been identified in Axenfeld-Rieger syndrome, a disorder characterized by anterior segment dysgenesis. We show that FOXC1 mutations also lead to corneal angiogenesis, and that mice homozygous for either a global (Foxc1

To identify elements of the human alpha subunit gene necessary for cell-specific expression, we generated an array of block mutations spanning approximately 400 base pairs (bp) of promoter proximal region and examined them using transient transfection analysis in pituitary (alpha T3) and placental (BeWo) cell lines. Comparison of promoter activity in the two cell types revealed both common and unique elements required for transcription in pituitary and placenta. Two strong elements, the cyclic AMP response element (CRE) and the upstream regulatory element (URE), regulate expression of the alpha subunit gene in BeWo cells. In contrast, promoter activity in alpha T3 cells requires an array of weaker elements. These include the CREs, the URE, as well as two previously described elements, pituitary glycoprotein hormone basal element (PGBE) and gonadotrope-specific element (GSE), and two new elements we designated as the alpha basal elements 1 and 2 (alpha BE1 and alpha BE2). These new elements reside between -316 and -302 bp (alpha BE1) and -296 and -285 bp (alpha BE2) of the human alpha subunit promoter and bind distinct proteins designated alpha BP1 and alpha BP2, respectively. Southwestern blot analysis revealed that alpha BE1 specifically binds 54- and 56-kDa proteins. Additional studies disclosed several potential interactions between proteins that bind the CRE and proteins that occupy PGBE, alpha BE1, and alpha BE2, suggesting that gonadotrope-specific expression occurs through a unique composite regulatory element that includes components of the placenta-specific enhancer.

Gram-negative bacteria such as Escherichia coli have an inner membrane and an asymmetric outer membrane (OM) that together protect the cytoplasm and act as a highly selective permeability barrier. Lipopolysaccharide (LPS) is the major component of the outer leaflet of the OM and is essential for the survival of nearly all Gram-negative bacteria. Recent advances in understanding the proteins involved in the transport of LPS across the periplasm and into the outer leaflet of the OM include the identification of seven proteins suggested to comprise the LPS transport (Lpt) system. Crystal structures of the periplasmic Lpt protein LptA have recently been reported and show that LptA forms oligomers in either an end-to-end arrangement or a side-byside dimer. It is not known if LptA oligomers bridge the periplasm to form a large, connected protein complex or if monomeric LptA acts as a periplasmic shuttle to transport LPS across the periplasm. Therefore, the studies presented here focus specifically on the LptA protein and its oligomeric arrangement and concentration dependence in solution using experimental data from several biophysical approaches, including laser light scattering, crosslinking, and double electron electron resonance spectroscopy. The results of these complementary techniques clearly show that LptA readily associates into stable, end-to-end, rod-shaped oligomers even at relatively low local protein concentrations and that LptA forms a continuous array of higher order oligomeric end-to-end structures as a function of increasing protein concentration.

The lipopolysaccharide (LPS)-rich outer membrane (OM) is a unique feature of Gramnegative bacteria, and LPS transport across the inner membrane (IM) and through the periplasm is essential to the biogenesis and maintenance of the OM. LPS is transported across the periplasm to the outer leaflet of the OM by the LPS transport (Lpt) system, which in Escherichia coli is comprised of seven recently identified proteins, including LptA, LptC, LptDE, and LptFGB 2 . Structures of the periplasmic protein LptA and the soluble portion of the membrane-associated protein LptC have been solved and show these two proteins to be highly structurally homologous with unique folds. LptA has been shown to form concentration dependent oligomers that stack end-to-end. LptA and LptC have been shown to associate in vivo and are expected to form a similar proteinprotein interface to that found in the LptA dimer. In these studies, we disrupted LptA oligomerization by introducing two point mutations that removed a lysine and glutamine side chain from the C-terminal b-strand of LptA. This loss of oligomerization was characterized using EPR spectroscopy techniques and the affinity of the interaction between the mutant LptA protein and WT LptC was determined using EPR spectroscopy (K d 5 15 lM) and isothermal titration calorimetry (K d 5 14 lM). K d values were also measured by EPR spectroscopy for the interaction between LptC and WT LptA (4 lM) and for WT LptA oligomerization (29 lM). These data suggest that the affinity between LptA and LptC is stronger than the affinity for LptA oligomerization.