Peripheral infusion of rat bone marrow derived endothelial progenitor cells leads to homing in acute lung injury

Background

Bone marrow-derived progenitors for both epithelial and endothelial cells have been observed in the lung. Besides mature endothelial cells (EC) that compose the adult vasculature, endothelial progenitor cells (EPC) are supposed to be released from the bone marrow into the peripheral blood after stimulation by distinct inflammatory injuries. Homing of ex vivo generated bone marrow-derived EPC into the injured lung has not been investigated so far. We therefore tested the hypothesis whether homing of EPC in damaged lung tissue occurs after intravenous administration.

Methods

Ex vivo generated, characterized and cultivated rat bone marrow-derived EPC were investigated for proliferation and vasculogenic properties in vitro. EPC were tested for their homing in a left-sided rat lung transplant model mimicking a severe acute lung injury. EPC were transplanted into the host animal by peripheral administration into the femoral vein (10⁶ cells). Rats were sacrificed 1, 4 or 9 days after lung transplantation and homing of EPC was evaluated by fluorescence microscopy. EPC were tested further for their involvement in vasculogenesis processes occurring in subcutaneously applied Matrigel in transplanted animals.

Results

We demonstrate the integration of intravenously injected EPC into the tissue of the transplanted left lung suffering from acute lung injury. EPC were localized in vessel walls as well as in destructed lung tissue. Virtually no cells were found in the right lung or in other organs. However, few EPC were found in subcutaneous Matrigel in transplanted rats.

Conclusion

Transplanted EPC may play an important role in reestablishing the endothelial integrity in vessels after severe injury or at inflamatory sites and might further contribute to vascular repair or wound healing processes in severely damaged tissue. Therapeutic applications of EPC transplantation may ensue.     

Characterization of Angiotensin Converting Enzyme-2 (ACE2) in Human Urine

Angiotensin converting enzyme-2 (ACE2) is a recently described membrane-bound carboxypeptidase identified by its homology to ACE, the enzyme responsible for the formation of the potent vasoconstrictor angiotensin II (Ang II). ACE2 inactivates Ang II and is thus thought to act in a counter-regulatory fashion to ACE. ACE2 is highly expressed in epithelial cells of distal renal tubules, and recent evidence indicates that expression is increased in a range of renal diseases. A soluble form of ACE, generated by proteolytic cleavage of the membrane-bound form, has been shown to be present in urine; although evidence for a similar release of ACE2 has been reported in cell culture, it is not yet known whether this occurs in vivo. The present study has identified ACE2 in human urine, both by a sensitive fluorescence-based activity assay and by Western immunoblot. Levels of ACE2 were surprisingly higher than ACE, which may reflect preferential targeting of the enzyme to the luminal surface of the renal epithelium. Future studies will determine whether increased expression of ACE2 in renal diseases are reflected in higher urinary levels of this novel enzyme.Australian Peptide Conference Issue.     

The yeast FET5 gene encodes a FET3 -related multicopper oxidase implicated in iron transport

The yeast FET3 gene encodes an integral membrane multicopper oxidase required for high-affinity iron uptake. The FET4 gene encodes an Fe(II) transporter required for low-affinity uptake. To identify other yeast genes involved in iron uptake, we isolated genes that could, when overexpressed, suppress the iron-limited growth defect of a fet3 fet4 mutant. The FET5 gene was isolated in this screen and it encodes a multicopper oxidase closely related to Fet3p. Several observations indicate that Fet5p plays a role analogous to Fet3p in iron transport. Suppression of the fet3 fet4 mutant phenotype by FET5 overexpression required the putative FTR1 transporter subunit of the high-affinity system. Fet5p is an integral membrane protein whose oxidase domain is located on the cell surface or within an intracellular compartment. Oxidase activity measured in cells with altered levels of FET5 expression suggested that Fet5p is a functional oxidase. FET5 overexpression increased the rate of iron uptake by a novel uptake system. Finally, FET5 mRNA levels are regulated by iron and are increased in cells grown in iron-limited media. These results suggest that Fet5p normally plays a role in the transport of iron.     

Endo‐ and exo‐inulinases: Enzyme‐substrate interaction and rational immobilization

Three‐dimensional models of exoinulinase from Bacillus stearothermophilus and endoinulinase from Aspergillus niger were built up by means of homology modeling. The crystal structure of exoinulinase from Aspergillus awamori was used as a template, which is the sole structure of inulinase resolved so far. Docking and molecular dynamics simulations were performed to investigate the differences between the two inulinases in terms of substrate selectivity. The analysis of the structural differences between the two inulinases provided the basis for the explanation of their different regio‐selectivity and for the understanding of enzyme‐substrate interactions. Surface analysis was performed to point out structural features that can affect the efficiency of enzymes also after immobilization. The computational analysis of the three‐dimensional models proved to be an effective tool for acquiring information and allowed to formulate an optimal immobilized biocatalyst even more active that the native one, thus enabling the full exploitation of the catalytic potential of these enzymes. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Differential Phenotypes of Tissue-Infiltrating T Cells during Angiotensin II-Induced Hypertension in Mice

Hypertension remains the leading risk factor for cardiovascular disease (CVD). Experimental hypertension is associated with increased T cell infiltration into blood pressure-controlling organs, such as the aorta and kidney; importantly in absence of T cells of the adaptive immune system, experimental hypertension is significantly blunted. However, the function and phenotype of these T cell infiltrates remains speculative and undefined in the setting of hypertension. The current study compared T cell-derived cytokine and reactive oxygen species (ROS) production from normotensive and hypertensive mice. Splenic, blood, aortic, kidney and brain T cells were isolated from C57BL/6J mice following 14-day vehicle or angiotensin (Ang) II (0.7 mg/kg/day, s.c.) infusion. T cell infiltration was increased in aorta, kidney and brain from hypertensive mice. Cytokine analysis in stimulated T cells indicated an overall Th1 pro-inflammatory phenotype, but a similar proportion (flow cytometry) and quantity (cytometric bead array) of IFN-γ, TNF-α, IL-4 and IL-17 between vehicle- and Ang II- treated groups. Strikingly, elevated T cell-derived production of a chemokine, chemokine C-C motif ligand 2 (CCL2), was observed in aorta (∼6-fold) and kidney in response to Ang II, but not in brain, spleen or blood. Moreover, T cell-derived ROS production in aorta was elevated ∼3 -fold in Ang II-treated mice (n = 7; P<0.05). Ang II-induced hypertension does not affect the overall T cell cytokine profile, but enhanced T cell-derived ROS production and/or leukocyte recruitment due to elevated CCL2, and this effect may be further amplified with increased infiltration of T cells. We have identified a potential hypertension-specific T cell phenotype that may represent a functional contribution of T cells to the development of hypertension, and likely several other associated vascular disorders.     

High-level production and covalent immobilization of Providencia rettgeri penicillin G acylase (PAC) from recombinant Pichia pastoris for the development of a novel and stable biocatalyst of industrial applicability

A complete, integrated process for the production of an innovative formulation of penicillin G acylase from Providencia rettgeri(rPAC(P.rett))of industrial applicability is reported. In order to improve the yield of rPAC, the clone LN5.5, carrying four copies of pac gene integrated into the genome of Pichia pastoris, was constructed. The proteinase activity of the recombinant strain was reduced by knockout of the PEP4 gene encoding for proteinase A, resulting in an increased rPAC(P.rett) activity of approximately 40% (3.8 U/mL vs. 2.7 U/mL produced by LN5.5 in flask). A high cell density fermentation process was established with a 5-day methanol induction phase and a final PAC activity of up to 27 U/mL. A single step rPAC(P.rett) purification was also developed with an enzyme activity yield of approximately 95%. The novel features of the rPAC(P.rett) expressed in P.pastoris were fully exploited and emphasized through the covalent immobilization of rPAC(P.rett). The enzyme was immobilized on a series of structurally correlated methacrylic polymers, specifically designed and produced for optimizing rPAC(P.rett) performances in both hydrolytic and synthetic processes. Polymers presenting aminic functionalities were the most efficient, leading to formulations with higher activity and stability (half time stability >3 years and specific activity ranging from 237 to 477 U/g (dry) based on benzylpenicillin hydrolysis). The efficiency of the immobilized rPAC(P.rett) was finally evaluated by studying the kinetically controlled synthesis of beta-lactam antibiotics (cephalexin) and estimating the synthesis/hydrolysis ratio (S/H), which is a crucial parameter for the feasibility of the process.     

Host Tissue and Glycan Binding Specificities of Avian Viral Attachment Proteins Using Novel Avian Tissue Microarrays

The initial interaction between viral attachment proteins and the host cell is a critical determinant for the susceptibility of a host for a particular virus. To increase our understanding of avian pathogens and the susceptibility of poultry species, we developed novel avian tissue microarrays (TMAs). Tissue binding profiles of avian viral attachment proteins were studied by performing histochemistry on multi-species TMA, comprising of selected tissues from ten avian species, and single-species TMAs, grouping organ systems of each species together. The attachment pattern of the hemagglutinin protein was in line with the reported tropism of influenza virus H5N1, confirming the validity of TMAs in profiling the initial virus-host interaction. The previously believed chicken-specific coronavirus (CoV) M41 spike (S1) protein displayed a broad attachment pattern to respiratory tissues of various avian species, albeit with lower affinity than hemagglutinin, suggesting that other avian species might be susceptible for chicken CoV. When comparing tissue-specific binding patterns of various avian coronaviral S1 proteins on the single-species TMAs, chicken and partridge CoV S1 had predominant affinity for the trachea, while pigeon CoV S1 showed marked preference for lung of their respective hosts. Binding of all coronaviral S1 proteins was dependent on sialic acids; however, while chicken CoV S1 preferred sialic acids type I lactosamine (Gal(1-3)GlcNAc) over type II (Gal(1-4)GlcNAc), the fine glycan specificities of pigeon and partridge CoVs were different, as chicken CoV S1-specific sialylglycopolymers could not block their binding to tissues. Taken together, TMAs provide a novel platform in the field of infectious diseases to allow identification of binding specificities of viral attachment proteins and are helpful to gain insight into the susceptibility of host and organ for avian pathogens.     

Expression profiling of microRNA using oligo DNA arrays

After 12 years from its first application, microarray technology has become the reference technique to monitor gene expression of thousands of genes in the same experiment. In the past few years an increasing amount of evidence showed the importance of non-coding RNA (ncRNA) in different human diseases. The microRNAs (miRNAs) are one of the groups of ncRNA. They are small RNA fragments, 19-25 nucleotides long, with a main regulatory function on both protein coding genes and non-coding RNAs. The application of microarray platforms applied to miRNA profiling determined their deregulation in virtually all human diseases that have been studied. We previously developed a custom miRNA microarray platform, and here we describe the protocol we used to work with it including the oligo design strategy, the microarray printing protocol, the target-probe hybridization and the signal detection.     

Soluble and Catalytically Active Endothelin Converting Enzyme-1 is Present in Cerebrospinal Fluid of Subarachnoid Hemorrhage Patients

Endothelin converting Enzyme-1 (ECE-1) is essential for the production of Endothelin-1 (ET-1), which is associated with vasospasm following subarachnoid hemorrhage (SAH). We have previously demonstrated the presence of a catalytically active soluble form of ECE-1 in the media of endothelial cells. We aimed to determine if this form of ECE-1 exists in vivo, in cerebrospinal fluid (CSF) of SAH patients. We examined CSF taken from SAH subjects for the presence of soluble ECE-1 using a bradykinin based quenched fluorescent substrate assay. We obtained further confirmation by characterizing the CSF mediated cleavage products of BigET-1 and BigET_18–34 (6 μg/ml) using mass spectrometry. The specificity of cleavage was confirmed using the ECE-1 inhibitor {"type":"entrez-protein","attrs":{"text":"CGS35066","term_id":"877962710","term_text":"CGS35066"}}CGS35066 5nmol/L. SAH CSF samples had mean ECE-1 activity of 0.127 ± 0.037 μmols of substrate cleaved/μl of CSF/24 h. The C-terminal peptides generated upon the cleavage of BigET-1 and BigET_18–34 were detected 48 h after incubation of these substrates with CSF. Cleavage of these substrates was inhibited by {"type":"entrez-protein","attrs":{"text":"CGS35066","term_id":"877962710","term_text":"CGS35066"}}CGS35066. Results of Western blots also produced strong evidence for the presence of truncated soluble ECE-1 in CSF. These results strongly suggest the presence of a truncated but catalytically active form of ECE-1 in the CSF of SAH subjects. Further studies are necessary to determine the biological significance of soluble ECE-1 in CSF of SAH subjects, including an association with vasospasm after SAH.Endothelin-1 (ET-1)¹ is the most potent vasoconstrictor in the central nervous system. Elevated levels of ET-1 in cerebrospinal fluid (CSF) have been implicated in the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage (SAH) (1). However, it is not known whether the production of ET-1 within the CSF space contributes to the pathogenesis of vasospasm. ET-1 is produced upon the cleavage of its precursor BigET-1 by the highly specific metalloprotease Endothelin Converting Enzyme-1 (ECE-1). The rate-limiting step of ET-1 production is the expression and localization of ECE-1, whose catalytic activity is confined to the extracellular C-terminal domain. Previously, we have demonstrated that the catalytically active C terminus can be shed from the cell surface (2). This results in the release of a truncated but catalytically active form of ECE-1 into the extracellular milieu.Although the presence of a soluble form of ECE-1 has been demonstrated in vitro, it is yet to be shown in any human biological fluid. In this study, we have used a combination of mass spectrometry, Western blotting as well as quenched fluorescent substrate (QFS) based enzyme assays to demonstrate for the first time the presence of catalytically active, soluble form of ECE-1 in CSF of SAH subjects.