Tobias Haase

Fluorescent fusion proteins of soluble guanylyl cyclase indicate proximity of the heme nitric oxide domain and catalytic domain.

Background To examine the structural organisation of heterodimeric soluble guanylyl cyclase (sGC) Förster resonance energy transfer (FRET) was measured between fluorescent proteins fused to the amino- and carboxy-terminal ends of the sGC β1 and α subunits. Methodology/Principal Findings Cyan fluorescent protein (CFP) was used as FRET donor and yellow fluorescent protein (YFP) as FRET acceptor. After generation of recombinant baculovirus, fluorescent-tagged sGC subunits were co-expressed in Sf9 cells. Fluorescent variants of sGC were analyzed in vitro in cytosolic fractions by sensitized emission FRET. Co-expression of the amino-terminally tagged α subunits with the carboxy-terminally tagged β1 subunit resulted in an enzyme complex that showed a FRET efficiency of 10% similar to fluorescent proteins separated by a helix of only 48 amino acids. Because these findings indicated that the amino-terminus of the α subunits is close to the carboxy-terminus of the β1 subunit we constructed fusion proteins where both subunits are connected by a fluorescent protein. The resulting constructs were not only fluorescent, they also showed preserved enzyme activity and regulation by NO. Conclusions/Significance Based on the ability of an amino-terminal fragment of the β1 subunit to inhibit activity of an heterodimer consisting only of the catalytic domains (αcatβcat), Winger and Marletta (Biochemistry 2005, 44:4083–90) have proposed a direct interaction of the amino-terminal region of β1 with the catalytic domains. In support of such a concept of “trans” regulation of sGC activity by the H-NOX domains our results indicate that the domains within sGC are organized in a way that allows for direct interaction of the amino-terminal regulatory domains with the carboxy-terminal catalytic region. In addition, we constructed “fluorescent-conjoined” sGCs by fusion of the α amino-terminus to the β1 carboxy-terminus leading to a monomeric, fluorescent and functional enzyme complex. To our knowledge this represents the first example where a fluorescent protein links two different subunits of a higher ordered complex to yield a stoichometrically fixed functionally active monomer.

The Amino-Terminus of Nitric Oxide Sensitive Guanylyl Cyclase α1 Does Not Affect Dimerization but Influences Subcellular Localization

Background Nitric oxide sensitive guanylyl cyclase (NOsGC) is a heterodimeric enzyme formed by an α- and a β1-subunit. A splice variant (C-α1) of the α1-subunit, lacking at least the first 236 amino acids has been described by Sharina et al. 2008 and has been shown to be expressed in differentiating human embryonic cells. Wagner et al. 2005 have shown that the amino acids 61–128 of the α1-subunit are mandatory for quantitative heterodimerization implying that the C-α1-splice variant should lose its capacity to dimerize quantitatively. Methodology/Principal Findings In the current study we demonstrate preserved quantitative dimerization of the C-α1-splice by co-purification with the β1-subunit. In addition we used fluorescence resonance energy transfer (FRET) based on fluorescence lifetime imaging (FLIM) using fusion proteins of the β1-subunit and the α1-subunit or the C-α1 variant with ECFP or EYFP. Analysis of the respective combinations in HEK-293 cells showed that the fluorescence lifetime was significantly shorter (≈0.3 ns) for α1/β1 and C-α1/β1 than the negative control. In addition we show that lack of the amino-terminus in the α1 splice variant directs it to a more oxidized subcellular compartment. Conclusions/Significance We conclude that the amino-terminus of the α1-subunit is dispensable for dimerization in-vivo and ex-vivo, but influences the subcellular trafficking.

Nitric oxide sensitive guanylyl cyclase activity decreases during cerebral postnatal development because of a reduction in heterodimerization

Soluble guanylyl cyclase (sGC) is the major physiological receptor for nitric oxide (NO) throughout the central nervous system. Three different subunits form the α1/β1 and α2/β1 heterodimeric enzymes that catalyze the reaction of GTP to the second messenger cGMP. Both forms contain a prosthetic heme group which binds NO and mediates activation by NO. A number of studies have shown that NO/cGMP signaling plays a major role in neuronal cell differentiation during development of the central nervous system. In the present work, we studied regulation and expression of sGC in brain of rats during postnatal development using biochemical methods. We consistently observed a surprising decrease in cerebral NO sensitive enzyme activity in adult animals in spite of stable expression of sGC subunits. Total hemoprotein heme content was decreased in cerebrum of adult animals, likely because of an increase in heme oxygenase activity. But the loss of sGC activity was not simply because of heme loss in intact heterodimeric enzymes. This was shown by enzyme activity determinations with cinaciguat which can be used to test heme occupancy in intact heterodimers. A reduction in heterodimerization in cerebrum of adult animals was demonstrated by co‐precipitation analysis of sGC subunits. This explained the observed decrease in NO sensitive guanylyl cyclase activity in cerebrum of adult animals. We conclude that differing efficiencies in heterodimer formation may be an important reason for the lack of correlation between sGC protein expression and sGC activity that has been described previously. We suggest that heterodimerization of sGC is a regulated process that changes during cerebral postnatal development because of still unknown signaling mechanisms.

Influence of fibre diameter and orientation of electrospun copolyetheresterurethanes on smooth muscle and endothelial cell behaviour

Polymers exhibiting cell-selective effects represent an extensive research field with high relevance for biomedical applications e.g. in the cardiovascular field supporting re-endothelialization while suppressing smooth muscle cell overgrowth. Such an endothelial cell-selective effect could be recently demonstrated for a copolyetheresterurethane (PDC) containing biodegradable poly(p-dioxanone) and poly(ε-caprolactone) segments, which selectively enhanced the adhesion of human umbilical vein endothelial cells (HUVEC) while suppressing the attachment of smooth muscle cells (SMC). In this study we investigated the influence of the fibre orientation (random and aligned) and fibre diameter (2 μm and 500 nm) of electrospun PDC scaffolds on the adhesion, proliferation and apoptosis of HUVEC and SMC. Adhesion, viability and proliferation of HUVEC was diminished when the fibre diameter was reduced to a submicron scale, while the orientation of the microfibres did only slightly influence the cellular behaviour. In contrast, a submicron fibre diameter improved SMC viability. In conclusion, PDC scaffolds with micron-sized single fibres could be promising candidate materials for cell-selective stent coatings.

In vivo biocompatibility assessment of poly(ether imide) electrospun scaffolds

Poly(ether imide) (PEI), which can be chemically functionalized with biologically active ligands, has emerged as a potential biomaterial for medical implants. Electrospun PEI scaffolds have shown advantageous properties, such as enhanced endothelial cell adherence, proliferation and low platelet adhesion in in vitro experiments. In this study, the in vivo behaviour of electrospun PEI scaffolds and PEI films was examined in a murine subcutaneous implantation model. Electrospun PEI scaffolds and films were surgically implanted subcutaneously in the dorsae of mice. The surrounding subcutaneous tissue response was examined via histopathological examination at 7 and 28 days after implantation. No serious adverse events were observed for both types of PEI implants. The presence of macrophages or foreign body giant cells in the vicinity of the implants and the formation of a fibrous capsule indicated a normal foreign body reaction towards PEI films and scaffolds. Capsule thickness and inflammatory infiltration cells significantly decreased for PEI scaffolds during days 7–28 while remaining unchanged for PEI films. The infiltration of cells into the implant was observed for PEI scaffolds 7 days after implantation and remained stable until 28 days of implantation. Additionally some, but not all, PEI scaffold implants induced the formation of functional blood vessels in the vicinity of the implants. Conclusively, this study demonstrates the in vivo biocompatibility of PEI implants, with favourable properties of electrospun PEI scaffolds regarding tissue integration and wound healing. Copyright

Combinatorial G-CSF/AMD3100 Treatment in Cardiac Repair after Myocardial Infarction

Aims Several studies suggest that circulating bone marrow derived stem cells promote the regeneration of ischemic tissues. For hematopoietic stem cell transplantation combinatorial granulocyte-colony stimulating factor (G-CSF)/Plerixafor (AMD3100) administration was shown to enhance mobilization of bone marrow derived stem cells compared to G-CSF monotherapy. Here we tested the hypothesis whether combinatorial G-CSF/AMD3100 therapy has beneficial effects in cardiac recovery in a mouse model of myocardial infarction. Methods We analyzed the effect of single G-CSF (250 µg/kg/day) and combinatorial G-CSF/AMD3100 (100 µg/kg/day) treatment on cardiac morphology, vascularization, and hemodynamics 28 days after permanent ligation of the left anterior descending artery (LAD). G-CSF treatment started directly after induction of myocardial infarction (MI) for 3 consecutive days followed by a single AMD3100 application on day three after MI in the G-CSF/AMD3100 group. Cell mobilization was assessed by flow cytometry of blood samples drawn from tail vein on day 0, 7, and 14. Results Peripheral blood analysis 7 days after MI showed enhanced mobilization of white blood cells (WBC) and endothelial progenitor cells (EPC) upon G-CSF and combinatorial G-CSF/AMD3100 treatment. However, single or combinatorial treatment showed no improvement in survival, left ventricular function, and infarction size compared to the saline treated control group 28 days after MI. Furthermore, no differences in histology and vascularization of infarcted hearts could be observed. Conclusion Although the implemented treatment regimen caused no adverse effects, our data show that combinatorial G-CSF/AMD therapy does not promote myocardial regeneration after permanent LAD occlusion.

Heterodimerization with the β1 subunit directs the α2 subunit of nitric oxide-sensitive guanylyl cyclase to calcium-insensitive cell-cell contacts in HEK293 cells: Interaction with Lin7a

Nitric oxide-sensitive guanylyl cyclase is a heterodimeric enzyme consisting of an α and a β subunit. Two different α subunits (α1 and α2) give rise to two heterodimeric enzymes α1/β1 and α2/β1. Both coexist in a wide range of tissues including blood vessels and the lung, but expression of the α2/β1 form is generally much lower and approaches levels similar to the α1/β1 form in the brain only. In the present paper, we show that the α2/β1 form interacts with Lin7a in mouse brain synaptosomes based on co-precipitation analysis. In HEK293 cells, we found that the overexpressed α2/β1 form, but not the α1/β1 form is directed to calcium-insensitive cell-cell contacts. The isolated PDZ binding motif of an amino-terminally truncated α2 subunit was sufficient for cell-cell contact localization. For the full length α2 subunit with the PDZ binding motif this was only the case in the heterodimer configuration with the β1 subunit, but not as isolated α2 subunit. We conclude that the PDZ binding motif of the α2 subunit is only accessible in the heterodimer conformation of the mature nitric oxide-sensitive enzyme. Interaction with Lin7a, a small scaffold protein important for synaptic function and cell polarity, can direct this complex to nectin based cell-cell contacts via MPP3 in HEK293 cells. We conclude that heterodimerization is a prerequisite for further protein-protein interactions that direct the α2/β1 form to strategic sites of the cell membrane with adjacent neighbouring cells. Drugs increasing the nitric oxide-sensitivity of this specific form may be particularly effective.

Protective function of ahnak1 in vascular healing after wire injury

Aim: Vascular remodeling following injury substantially accounts for restenosis and adverse clinical outcomes. In this study, we investigated the role of the giant scaffold protein Ahnak1 in vascular healing after endothelial denudation of the murine femoral artery. Methods: The spatiotemporal expression pattern of Ahnak1 and Ahnak2 was examined using specific antibodies and real-time quantitative PCR. Following wire-mediated endothelial injury of Ahnak1-deficient mice and wild-type (WT) littermates, the processes of vascular healing were analyzed. Results: Ahnak1 and Ahnak2 showed a mutually exclusive vascular expression pattern, with Ahnak1 being expressed in the endothelium and Ahnak2 in the medial cells in naïve WT arteries. After injury, a marked increase of Ahnak1- and Ahnak2-positive cells at the lesion site became evident. Both proteins showed a strong upregulation in neointimal cells 14 days after injury. Ahnak1-deficient mice showed delayed vascular healing and dramatically impaired re-endothelialization that resulted in prolonged adverse vascular remodeling, when compared to the WT littermates. Conclusion: The large scaffold and adaptor proteins Ahnak1 and Ahnak2 exhibit differential expression patterns and functions in naïve and injured arteries. Ahnak1 plays a nonredundant protective role in vascular healing.

Conjoined NO-sensitive guanylyl cyclases

Background NO-sensitive guanylyl cyclases (NO-GC’s) that catalyze the reaction of GTP to the second messenger molecule cGMP are heterodimeric enzymes consisting of an a and a b1 subunit. The two prevalent isoforms in humans (a1/b1, a2/b1) are time-tested targets for drugs that release nitric oxide (NO) and new compounds that either sensitize the enzyme for activation by NO or activate the enzyme independently of NO. Homologous NO-GC subunits consist of an amino-terminal HNOX domain followed by a PASand a coiled coil domain and the carboxy-terminal catalytic domain.

Direct evidence for close proximity of catalytic and regulatory domains of heterodimeric sGC based on fluorescence resonance energy transfer

Methods The FRET donor, CFP, and FRET acceptor, YFP, were fused to aminoand carboxy-terminal ends of sGC subunits. After generation of recombinant baculovirus strains fluorescent tagged sGC subunits were co-expressed in Sf9cells. Fluorescent variants of sGC were analyzed in vitro in cytosolic fractions by sensitized emission FRET. In addition, fluorescent tagged sGC subunits were analyzed in vivo using confocal laser scanning microscopy and fluorescence lifetime imaging (FLIM) on an inverted microscope.