Correlation of 99mTc-DPD bone scintigraphy with histological amyloid load in patients with ATTR cardiac amyloidosis.

BACKGROUND: The significance of measuring 99mTc-labelled-3,3-diphosphono-1,2-propanodicarboxylic acid (99mTc-DPD) in transthyretin (ATTR) cardiac amyloidosis has not been adequately studied. This single-centre observational study evaluated the correlation between 99mTc-DPD scintigraphy and histological amyloid load in endomyocardial biopsy (EMB). METHODS: Twenty-eight patients with biopsy-proven ATTR amyloidosis and concomitantly available 99mTc-DPD scintigraphy were included. Visual Perugini scoring, and (semi-)quantitative analysis of cardiac 99mTc-DPD uptake by planar whole-body imaging and single photon emission computed tomography (SPECT/CT) using regions of interest (ROI) were performed. From this, heart-to-whole-body ratio (H/WB) and heart-to-contralateral-chest ratio (H/CL) were calculated. The histological amyloid load was quantified using two different staining methods. RESULTS: Increased cardiac tracer uptake was documented in all patients (planar: ROImean 129 ± 37 cps; SPECT/CT: ROImean 369 ± 142 cps). Histological amyloid load (19 ± 13%) significantly correlated with Perugini score (r = 0.69, p < .001) as well as with cardiac 99mTc-DPD uptake (planar: r = 0.64, p < .001; H/WB: r = 0.50, p = .014; SPECT/CT: r = 0.53, p = .008; H/CL: r = 0.43, p = .037) (results are shown for correlations with Congo Red-staining). CONCLUSION: In ATTR, cardiac 99mTc-DPD uptake significantly correlated with histological amyloid load in EMB. Further studies are needed to implement thresholds in cardiac 99mTc-DPD uptake measurements for risk stratification and guidance of therapy.

The angiogenic neuropeptide catestatin exerts beneficial effects on human coronary vascular cells and cardiomyocytes.

INTRODUCTION: Myocardial infarction (MI) induces irreversible tissue damage, eventually leading to heart failure. Exogenous induction of angiogenesis positively influences ventricular remodeling after MI. Recently, we could show that therapeutic angiogenesis by the neuropeptide catestatin (CST) restores perfusion in the mouse hind limb ischemia model by the induction of angio-, arterio- and vasculogenesis. Thus, we assumed that CST might exert beneficial effects on cardiac cells. METHODS/RESULTS: To test the effect of CST on cardiac angiogenesis in-vitro matrigel assays with human coronary artery endothelial cells (HCAEC) were performed. CST significantly mediated capillary like tube formation comparable to vascular endothelial growth factor (VEGF), which was used as positive control. Interestingly, blockade of bFGF resulted in abrogation of observed effects. Moreover, CST induced proliferation of HCAEC and human coronary artery smooth muscle cells (HCASMC) as determined by BrdU-incorporation. Similar to the matrigel assay blockade of bFGF attenuated the effect. Consistent with these findings western blot assays revealed a bFGF-dependent phosphorylation of extracellular-signal regulated kinase (ERK) 1/2 by CST in these cell lines. Finally, CST protected human cardiomyocytes in-vitro from apoptosis. CONCLUSION: CST might qualify as potential candidate for therapeutic angiogenesis in MI.

Toll-Like Receptor 3 Mediates Aortic Stenosis Through a Conserved Mechanism of Calcification.

BACKGROUND: Calcific aortic valve disease (CAVD) is characterized by a phenotypic switch of valvular interstitial cells to bone-forming cells. Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors at the interface between innate immunity and tissue repair. Type I interferons (IFNs) are not only crucial for an adequate antiviral response but also implicated in bone formation. We hypothesized that the accumulation of endogenous TLR3 ligands in the valvular leaflets may promote the generation of osteoblast-like cells through enhanced type I IFN signaling. METHODS: Human valvular interstitial cells isolated from aortic valves were challenged with mechanical strain or synthetic TLR3 agonists and analyzed for bone formation, gene expression profiles, and IFN signaling pathways. Different inhibitors were used to delineate the engaged signaling pathways. Moreover, we screened a variety of potential lipids and proteoglycans known to accumulate in CAVD lesions as potential TLR3 ligands. Ligand-receptor interactions were characterized by in silico modeling and verified through immunoprecipitation experiments. Biglycan (Bgn), Tlr3, and IFN-α/ß receptor alpha chain (Ifnar1)-deficient mice and a specific zebrafish model were used to study the implication of the biglycan (BGN)-TLR3-IFN axis in both CAVD and bone formation in vivo. Two large-scale cohorts (GERA [Genetic Epidemiology Research on Adult Health and Aging], n=55 192 with 3469 aortic stenosis cases; UK Biobank, n=257 231 with 2213 aortic stenosis cases) were examined for genetic variation at genes implicated in BGN-TLR3-IFN signaling associating with CAVD in humans. RESULTS: Here, we identify TLR3 as a central molecular regulator of calcification in valvular interstitial cells and unravel BGN as a new endogenous agonist of TLR3. Posttranslational BGN maturation by xylosyltransferase 1 (XYLT1) is required for TLR3 activation. Moreover, BGN induces the transdifferentiation of valvular interstitial cells into bone-forming osteoblasts through the TLR3-dependent induction of type I IFNs. It is intriguing that Bgn-/-, Tlr3-/-, and Ifnar1-/- mice are protected against CAVD and display impaired bone formation. Meta-analysis of 2 large-scale cohorts with >300 000 individuals reveals that genetic variation at loci relevant to the XYLT1-BGN-TLR3-interferon-α/ß receptor alpha chain (IFNAR) 1 pathway is associated with CAVD in humans. CONCLUSIONS: This study identifies the BGN-TLR3-IFNAR1 axis as an evolutionarily conserved pathway governing calcification of the aortic valve and reveals a potential therapeutic target to prevent CAVD.

NKNK: a New Essential Motif in the C-Terminal Domain of HIV-1 Group M Integrases.

Using coevolution network interference based on comparison of two phylogenetically distantly related isolates, one from the main group M and the other from the minor group O of HIV-1, we identify, in the C-terminal domain (CTD) of integrase, a new functional motif constituted by four noncontiguous amino acids (N222K240N254K273). Mutating the lysines abolishes integration through decreased 3' processing and inefficient nuclear import of reverse-transcribed genomes. Solution of the crystal structures of wild-type (wt) and mutated CTDs shows that the motif generates a positive surface potential that is important for integration. The number of charges in the motif appears more crucial than their position within the motif. Indeed, the positions of the K's could be permutated or additional K's could be inserted in the motif, generally without affecting integration per se Despite this potential genetic flexibility, the NKNK arrangement is strictly conserved in natural sequences, indicative of an effective purifying selection exerted at steps other than integration. Accordingly, reverse transcription was reduced even in the mutants that retained wt integration levels, indicating that specifically the wt sequence is optimal for carrying out the multiple functions that integrase exerts. We propose that the existence of several amino acid arrangements within the motif, with comparable efficiencies of integration per se, might have constituted an asset for the acquisition of additional functions during viral evolution.IMPORTANCE Intensive studies of HIV-1 have revealed its extraordinary ability to adapt to environmental and immunological challenges, an ability that is also at the basis of antiviral treatment escape. Here, by deconvoluting the different roles of the viral integrase in the various steps of the infectious cycle, we report how the existence of alternative equally efficient structural arrangements for carrying out one function opens up the possibility of adapting to the optimization of further functionalities exerted by the same protein. Such a property provides an asset to increase the efficiency of the infectious process. On the other hand, though, the identification of this new motif provides a potential target for interfering simultaneously with multiple functions of the protein.

Shock Wave Therapy Improves Cardiac Function in a Model of Chronic Ischemic Heart Failure: Evidence for a Mechanism Involving VEGF Signaling and the Extracellular Matrix.

Background Mechanical stimulation of acute ischemic myocardium by shock wave therapy ( SWT ) is known to improve cardiac function by induction of angiogenesis. However, SWT in chronic heart failure is poorly understood. We aimed to study whether mechanical stimulation upon SWT improves heart function in chronic ischemic heart failure by induction of angiogenesis and postnatal vasculogenesis and to dissect underlying mechanisms. Methods and Results SWT was applied in a mouse model of chronic myocardial ischemia. To study effects of SWT on postnatal vasculogenesis, wild-type mice received bone marrow transplantation from green fluorescence protein donor mice. Underlying mechanisms were elucidated in vitro in endothelial cells and murine aortic rings. Echocardiography and pressure/volume measurements revealed improved left ventricular ejection fraction, myocardial contractility, and diastolic function and decreased myocardial fibrosis after treatment. Concomitantly, numbers of capillaries and arterioles were increased. SWT resulted in enhanced expression of the chemoattractant stromal cell-derived factor 1 in ischemic myocardium and serum. Treatment induced recruitment of bone marrow-derived endothelial cells to the site of injury. In vitro, SWT resulted in endothelial cell proliferation, enhanced survival, and capillary sprouting. The effects were vascular endothelial growth factor receptor 2 and heparan sulfate proteoglycan dependent. Conclusions SWT positively affects heart function in chronic ischemic heart failure by induction of angiogenesis and postnatal vasculogenesis. SWT upregulated pivotal angiogenic and vasculogenic factors in the myocardium in vivo and induced proliferative and anti-apoptotic effects on endothelial cells in vitro. Mechanistically, these effects depend on vascular endothelial growth factor signaling and heparan sulfate proteoglycans. SWT is a promising treatment option for regeneration of ischemic myocardium.

Nanoparticular delivery system for a secretoneurin derivative induces angiogenesis in a hind limb ischemia model.

Common therapeutic strategies for peripheral arterial disease often fail to re-establish sufficient blood flow within legs and feet of patients for avoiding critical limb ischemia, what is characterized by a substantial risk for amputation. The neuropeptide secretoneurin induces angiogenesis in models of limb and myocardial ischemia and might be a promising tool in the treatment of patients without the option of revascularization therapy for severe ischemia. Within this manuscript, the biologically active part of secretoneurin was identified, modified by induction of a cysteine residue to gain higher stability against enzymatic degradation and further packed into S-protected thiolated chitosan nanoparticles, which enable intra-muscular application of secretoneurin. Secretoneurin nanoparticles restored blood flow in a mouse hind limb ischemia model within one week, whereas control particles did not. In vitro testing also revealed the angiogenic, antiapoptotic and proliferative effects of the new secretoneurin derivate, as tested in primary human umbilical vein endothelial cells. With the work from this study we provide a new promising tool for treatment of peripheral arterial disease.

Potent Sensitisation of Cancer Cells to Anticancer Drugs by a Quadruple Mutant of the Human Deoxycytidine Kinase.

Identifying enzymes that, once introduced in cancer cells, lead to an increased efficiency of treatment constitutes an important goal for biomedical applications. Using an original procedure whereby mutant genes are generated based on the use of conditional lentivector genome mobilisation, we recently described, for the first time, the identification of a human deoxycytidine kinase (dCK) mutant (G12) that sensitises a panel of cancer cell lines to treatment with the dCK analogue gemcitabine. Here, starting from the G12 variant itself, we generated a new library and identified a mutant (M36) that triggers even greater sensitisation to gemcitabine than G12. With respect to G12, M36 presents an additional mutation located in the region that constitutes the interface of the dCK dimer. The simple presence of this mutation halves both the IC50 and the proportion of residual cells resistant to the treatment. Furthermore, the use of vectors with self-inactivating LTRs leads to an increased sensitivity to treatment, a result compatible with a relief of the transcriptional interference exerted by the U3 promoter on the internal promoter that drives the expression of M36. Importantly, a remarkable effect is also observed in treatments with the anticancer compound cytarabine (AraC), for which a 10,000 fold decrease in IC50 occurred. By triggering the sensitisation of various cancer cell types with poor prognosis to two commonly used anticancer compounds M36 is a promising candidate for suicide gene approaches.

Low energy shock wave therapy induces angiogenesis in acute hind-limb ischemia via VEGF receptor 2 phosphorylation.

OBJECTIVES: Low energy shock waves have been shown to induce angiogenesis, improve left ventricular ejection fraction and decrease angina symptoms in patients suffering from chronic ischemic heart disease. Whether there is as well an effect in acute ischemia was not yet investigated. METHODS: Hind-limb ischemia was induced in 10-12 weeks old male C57/Bl6 wild-type mice by excision of the left femoral artery. Animals were randomly divided in a treatment group (SWT, 300 shock waves at 0.1 mJ/mm2, 5 Hz) and untreated controls (CTR), n = 10 per group. The treatment group received shock wave therapy immediately after surgery. RESULTS: Higher gene expression and protein levels of angiogenic factors VEGF-A and PlGF, as well as their receptors Flt-1 and KDR have been found. This resulted in significantly more vessels per high-power field in SWT compared to controls. Improvement of blood perfusion in treatment animals was confirmed by laser Doppler perfusion imaging. Receptor tyrosine kinase profiler revealed significant phosphorylation of VEGF receptor 2 as an underlying mechanism of action. The effect of VEGF signaling was abolished upon incubation with a VEGFR2 inhibitor indicating that the effect is indeed VEGFR 2 dependent. CONCLUSIONS: Low energy shock wave treatment induces angiogenesis in acute ischemia via VEGF receptor 2 stimulation and shows the same promising effects as known from chronic myocardial ischemia. It may therefore develop as an adjunct to the treatment armentarium of acute muscle ischemia in limbs and myocardium.

Topical secretoneurin gene therapy accelerates diabetic wound healing by interaction between heparan-sulfate proteoglycans and basic FGF.

Diabetic foot ulcers represent a therapeutic problem of high clinical relevance. Reduced vascular supply, neuropathy and diminished expression of growth factors strongly contribute to wound healing impairment in diabetes. Secretoneurin, an angiogenic neuropeptide, has been shown to improve tissue perfusion in different animal models by increasing the amount of vessels in affected areas. Therefore, topical secretoneurin gene therapy was tested in a full thickness wound healing model in diabetic db/db mice. Secretoneurin significantly accelerated wound closure in these mice and immunohistochemistry revealed higher capillary and arteriole density in the wounded area compared to control mice. In-vitro, the mechanism of action of secretoneurin on human dermal microvascular endothelial cells was evaluated in normal and diabetic cells. Secretoneurin shows positive effects on in vitro angiogenesis, proliferation and apoptosis of these cells in a basic fibroblast growth factor dependent manner. A small molecular weight inhibitor revealed fibroblast growth factor receptor 3 as the main receptor for secretoneurin mediated effects. Additionally, we could identify heparan-sulfates as important co-factor of secretoneurin induced binding of basic fibroblast growth factor to human dermal endothelial cells. We suggest topical secretoneurin plasmid therapy as new tool for delayed wound healing in patients suffering from diabetes.

High-resolution NMR analysis of the conformations of native and base analog substituted retroviral and LTR-retrotransposon PPT primers.

A purine-rich region of the plus-strand RNA genome of retroviruses and long terminal repeat (LTR)-containing retrotransposons, known as the polypurine tract (PPT), is resistant to hydrolysis by the RNase H domain of reverse transcriptase (RT) and ultimately serves as a primer for plus-strand DNA synthesis. The mechanisms underlying PPT resistance and selective processing remain largely unknown. Here, two RNA/DNA hybrids derived from the PPTs of HIV-1 and Ty3 were probed using high-resolution NMR for preexisting structural distortions in the absence of RT. The PPTs were selectively modified through base-pair changes or by incorporation of the thymine isostere, 2,4-difluoro-5-methylbenzene (dF), into the DNA strand. Although both wild-type (WT) and mutated hybrids adopted global A-form-like helical geometries, observed structural perturbations in the base-pair and dF-modified hybrids suggested that the PPT hybrids may function as structurally coupled domains.

A ribose sugar conformational switch in the LTR-retrotransposon Ty3 polypurine tract-containing RNA/DNA hybrid.

To probe structural features of a polypurine tract (PPT) that mediate its specific recognition and processing, a model 20 bp RNA/DNA hybrid duplex, which includes the full PPT sequence of the Saccharomyces cerevisiae LTR-retrotransposon Ty3, has been investigated using solution NMR spectroscopy. While homonuclear NOESY and DQF-COSY analyses indicate that this PPT-containing RNA/DNA hybrid adopts an overall A-form-like helical geometry, an unexpected sugar pucker switch has been detected for the ribose at position +1, relative to the cleavage site, on the RNA strand. A model of the conformational changes induced by the A- to B-type sugar pucker switch shows a significant change in the backbone trajectory of the RNA strand, which alters the presentation of backbone phosphate and 2' hydroxyl groups 3' of this residue. This observation implies that part of the mechanism governing RNase H fidelity may be through distortion of the RNA/DNA helix one base ahead of the scissile bond.

The chaperoning and assistance roles of the HIV-1 nucleocapsid protein in proviral DNA synthesis and maintenance.

In the following three sections, we will briefly review the seminal roles of the HIV-1 nucleocapsid protein p7 (NCp7) in the fate of the HIV-1 full length RNA from genomic RNA in a dimeric form to the proviral DNA. Emphasis will be given to the mechanisms of NC-directed assistance to the genomic RNA and reverse transcriptase (RT) in the course of proviral DNA synthesis and to DNA integrity at the end of the polymerization process, and to the NC-assisted repair and recombination reactions fueling the viability and variability of the virus.

The chaperoning and assistance roles of the HIV-1 nucleocapsid protein in proviral DNA synthesis and maintenance.

In the following three sections we will briefly review the seminal roles of the HIV-1 nucleocapsid protein NCp7 in the fate of the HIV-1 full length RNA from genomic RNA in a dimeric form to the proviral DNA. Emphasis will be given to the mechanisms of NC-directed assistance to the genomic RNA and reverse transcriptase in the course of proviral DNA synthesis and to DNA integrity at the end of the polymerization process, and to the NC-assisted repair and recombination reactions fueling the viability and variability of the virus.

Nonpolar thymine isosteres in the Ty3 polypurine tract DNA template modulate processing and provide a model for its recognition by Ty3 reverse transcriptase.

Despite diverging in sequence and size, the polypurine tract (PPT) primers of retroviruses and long terminal repeat-containing retrotransposons are accurately processed from (+) U3 RNA and DNA by their cognate reverse transcriptases (RTs). In this paper, we demonstrate that misalignment of the Ty3 retrotransposon RT on the human immunodeficiency virus-1 PPT induces imprecise removal of adjacent (+)-RNA and failure to release (+)-DNA from the primer. Based on these observations, we explored the structural basis of Ty3 PPT recognition by chemically synthesizing RNA/DNA hybrids whose (-)-DNA template was substituted with the non-hydrogen-bonding thymine isostere 2,4-difluoro-5-methylbenzene (F). We observed a consistent spatial correlation between the site of T --> F substitution and enhanced ribonuclease H (RNase H) activity approximately 12-13 bp downstream. In the most pronounced case, dual T --> F substitution at PPT positions -1/-2 redirects RNase H cleavage almost exclusively to the novel site. The structural features of this unusual base suggest that its insertion into the Ty3 PPT (-)-DNA template weakens the duplex, inducing a destabilization that is recognized by a structural element of Ty3 RT approximately 12-13 bp from its RNase H catalytic center. A likely candidate for this interaction is the thumb subdomain, whose minor groove binding tract most likely contacts the duplex. The spatial relationship derived from T --> F substitution also infers that Ty3 PPT processing requires recognition of sequences in its immediate 5' vicinity, thereby locating the RNase H catalytic center over the PPT-U3 junction, a notion strengthened by additional mutagenesis studies of this paper.

Mutating conserved residues in the ribonuclease H domain of Ty3 reverse transcriptase affects specialized cleavage events.

The reverse transcriptase-associated ribonuclease H (RT/RNase H) domains from the gypsy group of retrotransposons, of which Ty3 is a member, share considerable sequence homology with their retroviral counterparts. However, the gypsy elements have a conserved tyrosine (position 459 in Ty3 RT) instead of the conserved histidine in the catalytic center of retroviral RTs such as at position 539 of HIV-1. In addition, the gypsy group shows conservation of histidine adjacent to the third of the metal-chelating carboxylate residues, which is Asp-426 of Ty3 RT. The role of these and additional catalytic residues was assessed with purified recombinant enzymes and through the ability of Ty3 mutants to support transposition in Saccaromyces cerevisiae. Although all mutations had minimal impact on DNA polymerase function, amidation of Asp-358, Glu-401, and Asp-426 eliminated Mg(2+)- and Mn(2+)-dependent RNase H function. Replacing His-427 and Tyr-459 with Ala and Asp-469 with Asn resulted in reduced RNase H activity in the presence of Mg(2+), whereas in the presence of Mn(2+) these mutants displayed a lack of turnover. Despite this, mutations at all positions were lethal for transposition. To reconcile these apparently contradictory findings, the efficiency of specialized RNase H-mediated events was examined for each enzyme. Mutants retaining RNase H activity on a heteropolymeric RNA.DNA hybrid failed to support DNA strand transfer and release of the (+) strand polypurine tract primer from (+) RNA, suggesting that interrupting one or both of these events might account for the transposition defect.

Altering the RNase H primer grip of human immunodeficiency virus reverse transcriptase modifies cleavage specificity.

Recent crystallographic data suggest that conserved residues in the connection subdomain and C-terminal ribonuclease H (RNase H) domain of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) contact the nascent DNA primer and modulate the trajectory of the template relative to the RNase H catalytic center. Within the RNase H domain, these residues include Thr473, Glu475, Lys476, Tyr501, and Ile505, while His539 and Asn474 interact with the scissile phosphate of the RNA template. Amino acid substitutions at several of these positions were evaluated in the context of hydrolysis of nonspecific RNA-DNA hybrids and substrates mimicking specific RNase H-mediated events. With the exception of mutant I505G, which exhibited a dimerization defect, substituting alanine at positions 473-476 and 501 had minimal consequences for DNA synthesis on duplex and hybrid DNA and RNA substrates. In contrast, the efficiency with which most mutants catalyzed polymerization-independent RNase H cleavage was sharply reduced. This deficiency was more pronounced when mutant enzymes were challenged to process the (+) strand polypurine tract (PPT) primer from either (+) RNA or a PPT/(+) DNA RNA/DNA chimera. Reduced polymerization-independent RNase H activity also significantly influenced the rate of DNA strand transfer, suggesting the donor template must be reduced in size below 13 nt before this event proceeds.