Regnase-1 overexpression as a therapeutic approach of Marfan syndrome.

Rupture or dissection of thoracic aortic aneurysms is still the leading cause of death for patients diagnosed with Marfan syndrome. Inflammation and matrix digestion regulated by matrix metalloproteases (MMPs) play a major role in the pathological remodeling of the aortic media. Regnase-1 is an endoribonuclease shown to cleave the mRNA of proinflammatory cytokines, such as interleukin-6. Considering the major anti-inflammatory effects of regnase-1, here, we aimed to determine whether adeno-associated virus (AAV)-mediated vascular overexpression of the protein could provide protection from the development and progression of aortic aneurysms in Marfan syndrome. The overexpression of regnase-1 resulted in a marked decrease in inflammatory parameters and elastin degradation in aortic smooth muscle cells in vitro. Intravenous injection of a vascular-targeted AAV vector resulted in the efficient transduction of the aortic wall and overexpression of regnase-1 in a murine model of Marfan syndrome, associated with lower circulating levels of proinflammatory cytokines and decreased MMP expression and activity. Regnase-1 overexpression strongly improved elastin architecture in the media and reduced aortic diameter at distinct locations. Therefore, AAV-mediated regnase-1 overexpression may represent a novel gene therapy approach for inhibiting aortic aneurysms in Marfan syndrome.

Impact of the -1T>C single-nucleotide polymorphism of the CD40 gene on the development of endothelial dysfunction in a pro-diabetic microenvironment.

BACKGROUND AND AIMS: Hyperglycemia reinforces pro-inflammatory conditions that enhance CD40 expression in endothelial cells (EC). Thymine to cytosine transition (-1T > C) in the promoter of the CD40 gene (rs1883832) further increases the abundance of CD40 protein on the EC surface. This study examines potential associations of the -1T > C SNP of the CD40 gene with type 1 (T1D) or type 2 (T2D) diabetes. Moreover, it investigates the impact of a pro-inflammatory diabetic microenvironment on gene expression in human cultured umbilical vein EC (HUVEC) derived from CC- vs. TT-genotype donors. METHODS: Tetra-ARMS-PCR was used to compare genotype distribution in 252 patients with diabetes. Soluble CD40 ligand (sCD40L) and soluble CD40 receptor (sCD40) plasma levels were monitored using ELISA. RNA-sequencing was performed with sCD40L-stimulated CC- and TT-genotype HUVEC. Quantitative PCR, Western blot, multiplex-sandwich ELISA array, and immunocytochemistry were used to analyse changes in gene expression in these cells. RESULTS: Homozygosity for the C-allele was associated with a significant 4.3-fold higher odds of developing T2D as compared to individuals homozygous for the T-allele. Inflammation and endothelial-to-mesenchymal transition (EndMT) driving genes were upregulated in CC-genotype but downregulated in TT-genotype HUVEC when exposed to sCD40L. Expression of EndMT markers significantly increased while that of endothelial markers decreased in HUVEC following exposure to hyperglycemia, tumour necrosis factor-α and sCD40L. CONCLUSIONS: The -1T > C SNP of the CD40 gene is a risk factor for T2D. Depending on the genotype, it differentially affects gene expression in human cultured EC. CC-genotype HUVEC adopt a pro-inflammatory and intermediate EndMT-like phenotype in a pro-diabetic microenvironment.

Ectodomain Shedding by ADAM17 Increases the Release of Soluble CD40 from Human Endothelial Cells under Pro-Inflammatory Conditions.

BACKGROUND: Homozygosity for the C allele of the -1T>C single nucleotide polymorphism (SNP) of the CD40 gene (rs1883832) is associated with susceptibility to coronary heart disease (CHD), enhanced CD40 expression, and shedding. The disintegrin metalloprotease ADAM17 can cleave various cell surface proteins. This study investigates an association between ADAM17-mediated CD40 shedding and inflammation in CC genotype human endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVEC) carrying the CC genotype were stimulated with soluble CD40 ligand (sCD40L) or tumor necrosis factor-α (TNFα). Messenger RNA and protein expression were determined with standard methods. Levels of high sensitive c-reactive protein (hs-CRP), interleukin-6 (IL-6), and sCD40 in plasma samples from patients with CHD were assessed using ELISA. RESULTS: ADAM17 surface abundance was elevated following stimulation with CD40L and TNFα just as its regulator iRhom2. Inhibition of ADAM17 prevented TNFα-induced sCD40 and soluble vascular cell adhesion molecule-1 release into the conditioned medium and reinforced CD40 surface abundance. Secondary to inhibition of ADAM17, stimulation with CD40L or TNFα upregulated monocyte chemoattractant protein-1 mRNA and protein. Levels of sCD40 and the inflammatory biomarkers hs-CRP and IL-6 were positively correlated in the plasma of patients with CHD. CONCLUSIONS: We provide a mechanism by which membrane-bound CD40 is shed from the endothelial cell surface by ADAM17, boosting sCD40 formation and limiting downstream CD40 signaling. Soluble CD40 may represent a robust biomarker for CHD, especially in conjunction with homozygosity for the C allele of the -1T>C SNP of the CD40 gene.

Translational Medicine: Towards Gene Therapy of Marfan Syndrome.

Marfan syndrome (MFS) is one of the most common inherited disorders of connective tissue caused by mutations of the fibrillin-1 gene (FBN1). Vascular abnormalities, such as the enlargement of the aorta with the risk of life-threatening rupture are frequently observed. However, current treatment is limited and therapeutic options focus solely on symptomatic therapy. Gene therapy focuses on genetically modifying cells to produce a therapeutic effect and may be a promising treatment option for MFS. Here, we first provide an overview of the historical background and characterization of MFS. Subsequently, we summarise current gene therapy options and possible translational concepts for this inherited disorder that affects connective tissue.

Inverse Regulation of Confluence-Dependent ADAMTS13 and von Willebrand Factor Expression in Human Endothelial Cells.

ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) is a zinc-containing metalloprotease also known as von Willebrand factor (vWF)-cleaving protease. Low ADAMTS13 plasma levels are associated with an increased risk of arterial thrombosis, including myocardial infarction and cerebrovascular disease. The expression and regulation of this metalloprotease in human endothelial cells have not been systematically investigated. In this study, we demonstrate that ADAMTS13 expression is inhibited by proinflammatory cytokines tumor necrosis factor-α and interferon-γ as well as by CD40 ligand, which was hitherto unknown. Factors protecting against atherosclerosis such as exposure to continuous unidirectional shear stress, interleukin-10, or different HMG-CoA reductase inhibitors like, e.g., simvastatin, atorvastatin, or rosuvastatin, did not influence ADAMTS13 expression. Unidirectional periodic orbital shear stress, mimicking oscillatory flow conditions found at atherosclerosis-prone arterial bifurcations, had also no effect. In contrast, a reciprocal correlation between ADAMTS13 and vWF expression in endothelial cells depending on the differentiation state was noted. ADAMTS13 abundance significantly rose on both the mRNA and intracellular protein level and also tethered to the endothelial glycocalyx with the degree of confluency while vWF protein levels were highest in proliferating cells but significantly decreased upon reaching confluence. This finding could explain the anti-inflammatory and antithrombotic phenotype of dormant endothelial cells mediated by contact inhibition.

AAV-mediated expression of NFAT decoy oligonucleotides protects from cardiac hypertrophy and heart failure.

Previous studies have underlined the substantial role of nuclear factor of activated T cells (NFAT) in hypertension-induced myocardial hypertrophy ultimately leading to heart failure. Here, we aimed at neutralizing four members of the NFAT family of transcription factors as a therapeutic strategy for myocardial hypertrophy transiting to heart failure through AAV-mediated cardiac expression of a RNA-based decoy oligonucleotide (dON) targeting NFATc1-c4. AAV-mediated dON expression markedly decreased endothelin-1 induced cardiomyocyte hypertrophy in vitro and resulted in efficient expression of these dONs in the heart of adult mice as evidenced by fluorescent in situ hybridization. Cardiomyocyte-specific dON expression both before and after induction of transverse aortic constriction protected mice from development of cardiac hypertrophy, cardiac remodeling, and heart failure. Singular systemic administration of AAVs enabling a cell-specific expression of dONs for selective neutralization of a given transcription factor may thus represent a novel and powerful therapeutic approach.

Alginate hydrogel polymers enable efficient delivery of a vascular-targeted AAV vector into aortic tissue.

Gene therapeutic approaches to aortic diseases require efficient vectors and delivery systems for transduction of endothelial cells (ECs) and smooth muscle cells (SMCs). Here, we developed a novel strategy to efficiently deliver a previously described vascular-specific adeno-associated viral (AAV) vector to the abdominal aorta by application of alginate hydrogels. To efficiently transduce ECs and SMCs, we used AAV9 vectors with a modified capsid (AAV9SLR) encoding enhanced green fluorescent protein (EGFP), as wild-type AAV vectors do not transduce ECs and SMCs well. AAV9SLR vectors were embedded into a solution containing sodium alginate and polymerized into hydrogels. Gels were surgically implanted around the adventitia of the infrarenal abdominal aorta of adult mice. Three weeks after surgery, an almost complete transduction of both the endothelium and tunica media adjacent to the gel was demonstrated in tissue sections. Hydrogel-mediated delivery resulted in induction of neutralizing antibodies but did not cause inflammatory responses in serum or the aortic wall. To further determine the translational potential, aortic tissue from patients was embedded ex vivo into AAV9SLR-containing hydrogel, and efficient transduction could be confirmed. These findings demonstrate that alginate hydrogel harboring a vascular-targeting AAV9SLR vector allows efficient local transduction of the aortic wall.

AAV-mediated AP-1 decoy oligonucleotide expression inhibits aortic elastolysis in a mouse model of Marfan syndrome.

AIMS: Marfan syndrome is one of the most common inherited disorders of connective tissue caused by fibrillin-1 mutations, characterized by enhanced transcription factor AP-1 DNA binding activity and subsequently abnormally increased expression and activity of matrix-metalloproteinases (MMPs). We aimed to establish a novel adeno-associated virus (AAV)-based strategy for long-term expression of an AP-1 neutralizing RNA hairpin (hp) decoy oligonucleotide (dON) in the aorta to prevent aortic elastolysis in a murine model of Marfan syndrome. METHODS AND RESULTS: Using fibrillin-1 hypomorphic mice (mgR/mgR), aortic grafts from young (9 weeks old) donor mgR/mgR mice were transduced ex vivo with AAV vectors and implanted as infrarenal aortic interposition grafts in mgR/mgR mice. Grafts were explanted after 30 days. For in vitro studies, isolated primary aortic smooth muscle cells (SMCs) from mgR/mgR mice were used. Elastica-van-Giesson staining visualized elastolysis, reactive oxygen species (ROS) production was assessed using dihydroethidine staining. RNA F.I.S.H. verified AP-1 hp dON generation in the ex vivo transduced aortic tissue. MMP expression and activity were assessed by western blotting and immunoprecipitation combined with zymography.Transduction resulted in stable therapeutic dON expression in endothelial and SMCs. MMP expression and activity, ROS formation as well as expression of monocyte chemoattractant protein-1 were significantly reduced. Monocyte graft infiltration declined and the integrity of the elastin architecture was maintained. RNAseq analysis confirmed the beneficial effect of AP-1 neutralization on the pro-inflammatory environment in SMCs. CONCLUSION: This novel approach protects from deterioration of aortic stability by sustained delivery of nucleic acids-based therapeutics and further elucidated how to interfere with the mechanism of elastolysis.

Endothelial CD40 Mediates Microvascular von Willebrand Factor-Dependent Platelet Adhesion Inducing Inflammatory Venothrombosis in ADAMTS13 Knockout Mice.

BACKGROUND: von Willebrand factor (vWF) plays an important role in platelet activation. CD40-CD40 ligand (CD40L) induced vWF release has been described in large vessels and cultured endothelium, but its role in the microcirculation is not known. Here, we studied whether CD40 is expressed in murine microvessels in vivo, whether CD40L induces platelet adhesion and leukocyte activation, and how deficiency of the vWF cleaving enzyme ADAMTS13 affects these processes. METHODS AND RESULTS: The role of CD40L in the formation of beaded platelet strings reflecting their adhesion to ultralarge vWF fibers (ULVWF) was analyzed in the murine cremaster microcirculation in vivo. Expression of CD40 and vWF was studied by immunohistochemistry in isolated and fixed cremasters. Microvascular CD40 was only expressed under inflammatory conditions and exclusively in venous endothelium. We demonstrate that CD40L treatment augmented the number of platelet strings, reflecting ULVWF multimer formation exclusively in venules and small veins. In ADAMTS13 knockout mice, the number of platelet strings further increased to a significant extent. As a consequence extensive thrombus formation was induced in venules of ADAMTS13 knockout mice. In addition, circulating leukocytes showed primary and rapid adherence to these platelet strings followed by preferential extravasation in these areas. CONCLUSION: CD40L is an important stimulus of microvascular endothelial ULVWF release, subsequent platelet string formation and leukocyte extravasation but only in venous vessels under inflammatory conditions. Here, the lack of ADAMTS13 leads to severe thrombus formation. The results identify CD40 expression and ADAMTS13 activity as important targets to prevent microvascular inflammatory thrombosis.

AAV-mediated TIMP-1 overexpression in aortic tissue reduces the severity of allograft vasculopathy in mice.

BACKGROUND: Allograft vasculopathy (AV) is the primary limiting factor for long-term graft survival. An increased activity of matrix metalloproteinases (MMPs) contributes to neointima formation in AV and represents a potential therapeutic target. Adeno-associated virus (AAV)-mediated gene therapy comprises a potentially benign vector model for the long-term expression of MMP antagonists. METHODS: Aortic allografts from DBA/2 mice were incubated with control buffer, AAV-enhanced green fluorescence protein (EGFP), or tissue inhibitor of metalloproteinases 1 (TIMP-1)-loaded AAV (AAV-TIMP-1) and transplanted into the infrarenal aorta of C57BL/6 mice. Cyclosporine A (10 mg/kg body weight) was administered daily. Explantation as well as histomorphometric and immunohistochemical evaluation was performed after 30 days. Matrix metalloproteinase (MMP) activity was visualized by gelatin in situ zymography. RESULTS: Intima-to-media area ratio and neointima formation were significantly reduced in the AAV-TIMP-1 treatment group compared with those in the control group (by 40%; p < 0.001) and the AAV-EGFP group (by 38.2%; p < 0.001). TIMP-1 overexpression positively affected several pathomechanisms for the development of AV both in vitro and in vivo as compared to that in the control groups: endothelium integrity was preserved as shown by zona occludens 1 and occludin staining; MMP9 expression and activity were significantly reduced (p = 0.01); and smooth muscle cell migration was significantly reduced as smooth muscle actin positive cells predominantly remained in the aortic media in the treatment group (p = 0.001). Moreover, macrophage infiltration was markedly reduced by 49% in the AAV-TIMP-1 group (p < 0.001). CONCLUSION: Immediate post-harvesting allograft incubation with AAV-TIMP-1 reduces neointima formation and macrophage infiltration, constituting a possible adjunct therapeutic strategy to preserve graft function after transplantation.

Functional association of a CD40 gene single-nucleotide polymorphism with the pathogenesis of coronary heart disease.

AIMS: Endothelial dysfunction is a major contributor to the pathogenesis of atherosclerosis. CD40-CD40 ligand interactions confer a pro-inflammatory phenotype to endothelial cells (ECs). Recently, a thymine to cytosine transition (-1T>C) in the Kozak sequence of the CD40 gene (rs1883832) has been associated with coronary heart disease (CHD) in an Asian population. As there are no reports yet regarding its role in other ethnic groups, this study determines if the -1T>C single-nucleotide polymorphism (SNP) could be a risk factor for CHD in Caucasians by performing an association study and elucidates its functional consequence in cultured ECs. METHODS AND RESULTS: Molecular and biochemical techniques, cell adhesion assays were used for genotype-stratified human EC characterization. SNP distribution in Caucasians was examined in a hospital-based case-control CHD study and serum levels of soluble CD40 (sCD40) were quantified by ELISA. The SNP in the CD40 gene affected baseline CD40 protein abundance on ECs. There was a genotype-dependent difference in CD40-mediated expression of pro-inflammatory genes. Monocyte adhesion was highest on the surface of cells homozygous for the C allele. Homozygosity for the C allele was associated with significant 2.32-fold higher odds of developing CHD as compared to TT genotype carriers. sCD40 plasma levels were genotype-dependently elevated in CHD patients, indicating a possible prognostic value. CONCLUSION: The C allele of the CD40 SNP provokes a pro-inflammatory EC phenotype, compensated by an enhanced CD40 shedding to neutralize excess CD40 ligand. Homozygosity for the C allele is the cause for a genetic susceptibility to atherosclerosis and its sequelae.

AAV-Mediated Expression of AP-1-Neutralizing RNA Decoy Oligonucleotides Attenuates Transplant Vasculopathy in Mouse Aortic Allografts.

Transplant vasculopathy (TV), characterized by obstructive lesions in affected vessels, represents one of the long-term complications of cardiac transplantation. Activation of the transcription factor activator protein-1 (AP-1) is implicated in smooth muscle cell (SMC) phenotypic switch from contractile to synthetic function, increasing the migration and proliferation rate of these cells. We hypothesize that adeno-associated virus (AAV)-mediated delivery of an RNA hairpin AP-1 decoy oligonucleotide (dON) might effectively ameliorate TV severity in a mouse aortic allograft model. Aortic allografts from DBA/2 mice ex vivo transduced with modified AAV9-SLR carrying a targeting peptide within the capsid surface were transplanted into the infrarenal aorta of C57BL/6 mice. Cyclosporine A (10 mg/kg BW) was administered daily. AP-1 dONs were intracellularly expressed in the graft tissue as small hairpin RNA proved by fluorescent in situ hybridization. Explantation after 30 days and histomorphometric evaluation revealed that AP-1 dON treatment significantly reduced intima-to-media ratio by 41.5% (p < 0.05) in the grafts. In addition, expression of adhesion molecules, cytokines, as well as numbers of proliferative SMCs, matrix metalloproteinase-9-positive cells, and inflammatory cell infiltration were significantly decreased in treated aortic grafts. Our findings demonstrate the feasibility, efficacy, and specificity of the anti-AP-1 RNA dON approach for the treatment of allograft vasculopathy in an animal model. Moreover, the AAV-based approach in general provides the possibility to achieve a prolonged delivery of nucleic-acids-based therapeutics in to the blood vessel wall.

Reduction of Transplant Vasculopathy by Intraoperative Nucleic Acid-based Therapy in a Mouse Aortic Allograft Model.

BACKGROUND: Transplant vasculopathy (TV) is the main limiting factor for long-term graft survival characterized by fibrosis, myofibroblast, and smooth muscle cell (SMC) proliferation. Decoy oligodeoxynucleotide (dODN) against the transcription factor activator protein-1 (AP-1) might interfere with the expression of AV-related genes that govern neointima formation. METHODS: Aortic allografts from DBA/2 mice were incubated with control buffer, consensus, or mutated control AP-1 dODN and were transplanted into the infrarenal aorta of C57BL/6 mice. Cyclosporine A (10 mg/kg body weight [BW]) was administered daily. Explantation and histomorphometric and immunohistochemical evaluation was performed after 30 days. Matrix metalloproteinase (MMP) activity was visualized by gelatin in situ zymography. RESULTS: Intima-to-media (I/M) ratio and neointima formation were significantly reduced in the consensus AP-1 dODN treatment group by 37% (p < 0.05) and 67% (p < 0.01), respectively. SMC α-actin-2 staining and macrophage marker expression revealed a marked reduction in the neointima. I/M ratio was found to correlate with the number of tissue macrophages (p < 0.05). MMP and fibrosis marker expression were not significantly altered. CONCLUSION: Intraoperative AP-1dODN utilization might be a strategy to preserve graft function after transplantation.

Strategy for marker-based differentiation of pro- and anti-inflammatory macrophages using matrix-assisted laser desorption/ionization mass spectrometry imaging.

Macrophages are large phagocytes playing a crucial role in the development and progression of atherosclerosis. The phenotypic polarization and activation of macrophages in atherosclerotic plaques depends on their complex micro-environment and at the same time has a major impact on the vulnerability or stability of advanced atherosclerotic lesions. Many in vitro and in vivo studies have been designed to define markers for macrophage subtypes to better understand the mechanism of plaque progression but they have rather added to the confusion. Nonetheless, some of the in vitro defined macrophage subtypes, like the pro-inflammatory M1 or the anti-inflammatory M2a/b/c macrophage, have been shown to be present in atherosclerotic plaques. Herein, we developed a comprehensive workflow to distinguish between human in vitro differentiated pro-inflammatory M1 and anti-inflammatory M2a and M2c macrophages. The cells were analyzed using qPCR and FACS analyses for defining suitable markers on the transcript (mRNA) and protein level as well as MALDI MSI for the assignment of metabolic markers, which can be used for the identification of the corresponding macrophage subtypes in atherosclerotic plaques. Data obtained using both qPCR and FACS analyses were in agreement with the literature. For the analysis of the macrophages with MALDI MSI, a comprehensive workflow was developed and the obtained data were subjected to different statistical analysis methods like principal component analysis (PCA) to define markers for each macrophage type. Our MALDI MSI results revealed that the method produces reliable and reproducible results but that the heterogeneity of the monocytes derived from different donors is too high to define universal markers on the metabolic level. Moreover, the results show that a sample set of three biological replicates is not sufficient to obtain representative data and therefore we recommend performing ring experiments in which the samples are measured by different laboratories.

Role of CD40 and ADAMTS13 in von Willebrand factor-mediated endothelial cell-platelet-monocyte interaction.

Monocyte extravasation into the vessel wall is a key step in atherogenesis. It is still elusive how monocytes transmigrate through the endothelial cell (EC) monolayer at atherosclerosis predilection sites. Platelets tethered to ultra-large von Willebrand factor (ULVWF) multimers deposited on the luminal EC surface following CD40 ligand (CD154) stimulation may facilitate monocyte diapedesis. Human ECs grown in a parallel plate flow chamber for live-cell imaging or Transwell permeable supports for transmigration assay were exposed to fluid or orbital shear stress and CD154. Human isolated platelets and/or monocytes were superfused over or added on top of the EC monolayer. Plasma levels and activity of the ULVWF multimer-cleaving protease ADAMTS13 were compared between coronary artery disease (CAD) patients and controls and were verified by the bioassay. Two-photon intravital microscopy was performed to monitor CD154-dependent leukocyte recruitment in the cremaster microcirculation of ADAMTS13-deficient versus wild-type mice. CD154-induced ULVWF multimer-platelet string formation on the EC surface trapped monocytes and facilitated transmigration through the EC monolayer despite high shear stress. Two-photon intravital microscopy revealed CD154-induced ULVWF multimer-platelet string formation preferentially in venules, due to strong EC expression of CD40, causing prominent downstream leukocyte extravasation. Plasma ADAMTS13 abundance and activity were significantly reduced in CAD patients and strongly facilitated both ULVWF multimer-platelet string formation and monocyte trapping in vitro. Moderate ADAMTS13 deficiency in CAD patients augments CD154-mediated deposition of platelet-decorated ULVWF multimers on the luminal EC surface, reinforcing the trapping of circulating monocytes at atherosclerosis predilection sites and promoting their diapedesis.

Carnosine Catalyzes the Formation of the Oligo/Polymeric Products of Methylglyoxal.

BACKGROUND/AIMS: Reactive dicarbonyl compounds, such as methylglyoxal (MG), contribute to diabetic complications. MG-scavenging capacities of carnosine and anserine, which have been shown to mitigate diabetic nephropathy, were evaluated in vitro and in vivo. METHODS: MG-induced cell toxicity was characterized by MTT and MG-H1-formation, scavenging abilities by Western Blot and NMR spectroscopies, cellular carnosine transport by qPCR and microplate luminescence and carnosine concentration by HPLC. RESULTS: In vitro, carnosine and anserine dose-dependently reduced N-carboxyethyl lysine (CEL) and advanced glycation end products (AGEs) formation. NMR studies revealed the formation of oligo/polymeric products of MG catalyzed by carnosine or anserine. MG toxicity (0.3-1 mM) was dose-dependent for podocytes, tubular and mesangial cells whereas low MG levels (0.2 mM) resulted in increased cell viability in podocytes (143±13%, p<0.001) and tubular cells (129±3%, p<0.001). Incubation with carnosine/anserine did not reduce MG-induced toxicity, independent of incubation times and across large ranges of MG to carnosine/anserine ratios. Cellular carnosine uptake was low (<0.1% in 20 hours) and cellular carnosine concentrations remained unaffected. The putative carnosine transporter PHT1 along with the taurine transporter (TauT) was expressed in all cell types while PEPT1, PEPT2 and PHT2, also belonging to the proton-coupled oligopeptide transporter (POT) family, were only expressed in tubular cells. CONCLUSION: While carnosine and anserine catalyze the formation of MG oligo/polymers, the molar ratios required for protection from MG-induced cellular toxicity are not achievable in renal cells. The effect of carnosine in vivo, to mitigate diabetic nephropathy may therefore be independent upon its ability to scavenge MG and/or carnosine is mainly acting extracellularly.

Allosteric inhibition of carnosinase (CN1) by inducing a conformational shift.

In humans, low serum carnosinase (CN1) activity protects patients with type 2 diabetes from diabetic nephropathy. We now characterized the interaction of thiol-containing compounds with CN1 cysteine residue at position 102, which is important for CN1 activity. Reduced glutathione (GSH), N-acetylcysteine and cysteine (3.2 ± 0.4, 2.0 ± 0.3, 1.6 ± 0.2 µmol/mg/h/mM; p < .05) lowered dose-dependently recombinant CN1 (rCN1) efficiency (5.2 ± 0.2 µmol/mg/h/mM) and normalized increased CN1 activity renal tissue samples of diabetic mice. Inhibition was allosteric. Substitution of rCN1 cysteine residues at position 102 (Mut1C102S) and 229 (Mut2C229S) revealed that only cysteine-102 is influenced by cysteinylation. Molecular dynamic simulation confirmed a conformational rearrangement of negatively charged residues surrounding the zinc ions causing a partial shift of the carnosine ammonium head and resulting in a less effective pose of the substrate within the catalytic cavity and decreased activity. Cysteine-compounds influence the dynamic behaviour of CN1 and therefore present a promising option for the treatment of diabetes.

Transcription factor decoy technology: A therapeutic update.

Targeting transcription factors represents one possibility to interfere with a known activated regulatory pathway that promotes disease. Double-stranded transcription factor decoy (TFD) oligodeoxynucleotides (ODN) are therapeutic drug candidates, which are able to specifically target and neutralize key transcription factors involved in the pathogenesis of a given disease. These short double-stranded TFD molecules mimic the consensus DNA binding site of a specific transcription factor in the promoter region of its target genes. Therefore, it is possible to exploit this nucleic acid-based drug class for the treatment of diseases caused by aberrant expression of such target genes the products of which are involved in disease initiation and progression. This research update focuses firstly on the mechanism of action of TFD molecules. Long-term effects of such ODNs depend on their stability and the efficiency by which they are delivered to the target tissue and taken up by their target cells. Hence structural modifications like e.g., single-stranded TFD molecules hybridising to itself to form an intramolecular hairpin molecule or circular ODNs assuming a dumbbell configuration, intended to enhance both stability and efficacy, are addressed. Also specific drug delivery methods like ultrasound-targeted microbubble destruction with TFD ODN-coated microbubbles or adeno-associated viral (AAV) vectors for tissue-specific transduction and long-term TFD molecule expression in non-dividing cells will be discussed. Finally, current therapeutic applications of TFD ODN will be summarized.

Loss of Endothelial Barrier in Marfan Mice (mgR/mgR) Results in Severe Inflammation after Adenoviral Gene Therapy.

OBJECTIVES: Marfan syndrome is an autosomal dominant inherited disorder of connective tissue. The vascular complications of Marfan syndrome have the biggest impact on life expectancy. The aorta of Marfan patients reveals degradation of elastin layers caused by increased proteolytic activity of matrix metalloproteinases (MMPs). In this study we performed adenoviral gene transfer of human tissue inhibitor of matrix metalloproteinases-1 (hTIMP-1) in aortic grafts of fibrillin-1 deficient Marfan mice (mgR/mgR) in order to reduce elastolysis. METHODS: We performed heterotopic infrarenal transplantation of the thoracic aorta in female mice (n = 7 per group). Before implantation, mgR/mgR and wild-type aortas (WT, C57BL/6) were transduced ex vivo with an adenoviral vector coding for human TIMP-1 (Ad.hTIMP-1) or ß-galactosidase (Ad.ß-Gal). As control mgR/mgR and wild-type aortas received no gene therapy. Thirty days after surgery, overexpression of the transgene was assessed by immunohistochemistry (IHC) and collagen in situ zymography. Histologic staining was performed to investigate inflammation, the neointimal index (NI), and elastin breaks. Endothelial barrier function of native not virus-exposed aortas was evaluated by perfusion of fluorescent albumin and examinations of virus-exposed tissue were performed by transmission electron microscopy (TEM). RESULTS: IHC and ISZ revealed sufficient expression of the transgene. Severe cellular inflammation and intima hyperplasia were seen only in adenovirus treated mgR/mgR aortas (Ad.ß-Gal, Ad.hTIMP-1 NI: 0.23; 0.43), but not in native and Ad.hTIMP-1 treated WT (NI: 0.01; 0.00). Compared to native mgR/mgR and Ad.hTIMP-1 treated WT aorta, the NI is highly significant greater in Ad.hTIMP-1 transduced mgR/mgR aorta (p = 0.001; p = 0.001). As expected, untreated Marfan grafts showed significant more elastolysis compared to WT (p = 0.001). However, elastolysis in Marfan aortas was not reduced by adenoviral overexpression of hTIMP-1 (compared to untreated Marfan aorta: Ad.hTIMP-1 p = 0.902; control Ad.ß-Gal. p = 0.165). The virus-untreated and not transplanted mgR/mgR aorta revealed a significant increase of albumin diffusion through the endothelial barrier (p = 0.037). TEM analysis of adenovirus-exposed mgR/mgR aortas displayed disruption of the basement membrane and basolateral space. CONCLUSIONS: Murine Marfan aortic grafts developed severe inflammation after adenoviral contact. We demonstrated that fibrillin-1 deficiency is associated with relevant dysfunction of the endothelial barrier that enables adenovirus to induce vessel-harming inflammation. Endothelial dysfunction may play a pivotal role in the development of the vascular phenotype of Marfan syndrome.

Varicose Remodeling of Veins Is Suppressed by 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors.

BACKGROUND: Despite the high prevalence of chronic venous insufficiency and varicose veins in the Western world, suitable pharmaceutical therapies for these venous diseases have not been explored to date. In this context, we recently reported that a chronic increase in venous wall stress or biomechanical stretch is sufficient to cause development of varicose veins through the activation of the transcription factor activator protein 1. METHODS AND RESULTS: We investigated whether deleterious venous remodeling is suppressed by the pleiotropic effects of statins. In vitro, activator protein 1 activity was inhibited by two 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, rosuvastatin and atorvastatin, in stretch-stimulated human venous smooth muscle cells. Correspondingly, both statins inhibited venous smooth muscle cell proliferation as well as mRNA expression of the activator protein 1 target gene monocyte chemotactic protein 1 (MCP1). In isolated mouse veins exposed to an increased level of intraluminal pressure, statin treatment diminished proliferation of venous smooth muscle cells and protein abundance of MCP1 while suppressing the development of varicose veins in a corresponding animal model by almost 80%. Further analyses of human varicose vein samples from patients chronically treated with statins indicated a decrease in venous smooth muscle cell proliferation and MCP1 abundance compared with samples from untreated patients. CONCLUSIONS: Our findings imply that both atorvastatin and rosuvastatin effectively inhibit the development of varicose veins, at least partially, by interfering with wall stress-mediated activator protein 1 activity in venous smooth muscle cells. For the first time, this study reveals a potential pharmacological treatment option that may be suitable to prevent growth of varicose veins and to limit formation of recurrence after varicose vein surgery.