Type 1 diabetes is associated with reduced vascular repair, as indicated by impaired wound healing and reduced collateral formation in ischemia. Recently, endothelial progenitor cells (EPCs) have been identified as important regulators of these processes. We therefore explored the concept that EPCs are dysfunctional in diabetes. The number of EPCs obtained from type 1 diabetic patients in culture was 44% lower compared with age- and sex-matched control subjects (P < 0.001). This reduction was inversely related to levels of HbA_1c (R = −0.68, P = 0.01). In addition, we demonstrated that patient EPCs were also impaired in function using an in vitro angiogenesis assay. Conditioned media from patient EPCs were significantly reduced in their capacity to support endothelial tube formation in comparison to control EPCs. Therefore, despite culturing the EPCs under normoglycemic conditions, functional differences between patient and control EPCs were maintained. Our findings demonstrate that adverse metabolic stress factors in type 1 diabetes are associated with reduced EPC numbers and angiogenicity. We hypothesize that EPC dysfunction contributes to the pathogenesis of vascular complications in type 1 diabetes.

We have recently observed that chitotriosidase, a chitinolytic enzyme, is secreted by activated human macrophages and is markedly elevated in plasma of Gaucher disease patients (Hollak, C. E. M., van Weely, S., van Oers, M. H. J., and Aerts, J. M. F. G. (1994) J. Clin. Invest. 93, 1288-1292). Here, we report on the cloning of the corresponding cDNA. The nucleotide sequence of the cloned cDNA predicts a protein with amino acid sequences identical to those established for purified chitotriosidase. Secretion of active chitotriosidase was obtained after transient transfection of COS-1 cells with the cloned cDNA, confirming its identity as chitotriosidase cDNA. Chitotriosidase contains several regions with high homology to those present in chitinases from different species belonging to family 18 of glycosyl hydrolases. Northern blot analysis shows that expression of chitotriosidase mRNA occurs only at a late stage of differentiation of monocytes to activated macrophages in culture. Our results show that, in contrast to previous beliefs, human macrophages can synthesize a functional chitinase, a highly conserved enzyme with a strongly regulated expression. This enzyme may play a role in the degradation of chitin-containing pathogens and can be used as a marker for specific disease states.

We describe a genome-wide characterization of mRNA transcript levels in yeast grown on the fatty acid oleate, determined using Serial Analysis of Gene Expression (SAGE). Comparison of this SAGE library with that reported for glucose grown cells revealed the dramatic adaptive response of yeast to a change in carbon source. A major fraction (>20%) of the 15,000 mRNA molecules in a yeast cell comprised differentially expressed transcripts, which were derived from only 2% of the total number of ∼6300 yeast genes. Most of the mRNAs that were differentially expressed code for enzymes or for other proteins participating in metabolism (e.g., metabolite transporters). In oleate-grown cells, this was exemplified by the huge increase of mRNAs encoding the peroxisomal β-oxidation enzymes required for degradation of fatty acids. The data provide evidence for the existence of redox shuttles across organellar membranes that involve peroxisomal, cytoplasmic, and mitochondrial enzymes. We also analyzed the mRNA profile of a mutant strain with deletions of the PIP2and OAF1 genes, encoding transcription factors required for induction of genes encoding peroxisomal proteins. Induction of genes under the immediate control of these factors was abolished; other genes were up-regulated, indicating an adaptive response to the changed metabolism imposed by the genetic impairment. We describe a statistical method for analysis of data obtained by SAGE.

MicroRNAs are negative regulators of gene expression that play a key role in cell‐type specific differentiation and modulation of cell function and have been proposed to be involved in neovascularization. Previously, using an extensive cloning and sequencing approach, we identified miR‐126 to be specifically and highly expressed in human endothelial cells (EC). Here, we demonstrate EC‐specific expression of miR‐126 in capillaries and the larger vessels in vivo. We therefore explored the potential role of miR‐126 in arteriogenesis and angiogenesis. Using miR‐reporter constructs, we show that miR‐126 is functionally active in EC in vitro and that it could be specifically repressed using antagomirs specifically targeting miR‐126. To study the consequences of miR‐126 silencing on vascular regeneration, mice were injected with a single dose of antagomir‐126 or a control ‘scramblemir’ and exposed to ischemia of the left hindlimb by ligation of the femoral artery. Although miR‐126 was effectively silenced in mice treated with a single, high dose (HD) of antagomir‐126, laser Doppler perfusion imaging did not show effects on blood flow recovery. In contrast, quantification of the capillary density in the gastrocnemius muscle revealed that mice treated with a HD of antagomir‐126 had a markedly reduced angiogenic response. Aortic explant cultures of the mice confirmed the role of miR‐126 in angiogenesis. Our data demonstrate a facilitary function for miR‐126 in ischemia‐induced angiogenesis and show the efficacy and specificity of antagomir‐induced silencing of EC‐specific microRNAs in vivo.

IcroRNAs are 20- to 23-nucleotide RNA molecules that can regulate gene expression. Currently >400 microRNAs have been experimentally identified in mammalian genomes, whereas estimates go up to 1000 and beyond. Here we show that many more mammalian microRNAs exist. We discovered novel microRNA candidates using two approaches: testing of computationally predicted microRNAs by a modified microarray-based detection system, and cloning and sequencing of large numbers of small RNAs from different human and mouse tissues. Together these efforts experimentally identified 348 novel mouse and 81 novel human microRNA candidate genes. Most novel microRNAs candidates are not conserved beyond mammals, and ~10% are taxon-specific. Our analyses indicate that the entire microRNA repertoire is not remotely exhausted.

In the present study, the interaction between the endocytic receptor low density lipoprotein receptor-related protein (LRP) and coagulation factor VIII (FVIII) was investigated. Using purified components, FVIII was found to bind to LRP in a reversible and dose-dependent manner (K _d ≈ 60 nm). The interaction appeared to be specific because the LRP antagonist receptor-associated protein readily inhibited binding of FVIII to LRP (IC₅₀ ≈ 1 nm). In addition, a 12-fold molar excess of the physiological carrier of FVIII,i.e. von Willebrand factor (vWF), reduced the binding of FVIII to LRP by over 90%. Cellular degradation of¹²⁵I-labeled FVIII by LRP-expressing cells (≈ 8 fmol/10⁵ cells after a 4.5-h incubation) was reduced by approximately 70% in the presence of receptor-associated protein. LRP-directed antibodies inhibited degradation to a similar extent, indicating that LRP indeed contributes to binding and transport of FVIII to the intracellular degradation pathway. Degradation of FVIII was completely inhibited by vWF. Because vWF binding by FVIII involves its light chain, LRP binding to this subunit was studied. In ligand blotting experiments, binding of FVIII light chain to LRP could be visualized. More detailed analysis revealed that FVIII light chain interacts with LRP with moderate affinity (k _on≈ 5 × 10⁴ m ⁻¹s⁻¹; k _off ≈ 2.5 × 10⁻³ s⁻¹; K _d ≈ 50 nm). Furthermore, experiments using recombinant FVIII C2 domain showed that this domain contributes to the interaction with LRP. In contrast, no association of FVIII heavy chain to LRP could be detected under the same experimental conditions. Collectively, our data demonstrate that in vitro LRP is able to bind FVIII at the cell surface and to mediate its transport to the intracellular degradation pathway. FVIII-LRP interaction involves the FVIII light chain, and FVIII-vWF complex formation plays a regulatory role in LRP binding. Our findings may explain the beneficial effect of vWF on thein vivo survival of FVIII.