Paul H.A. Quax

Replication-Deficient Human Adenovirus Type 35 Vectors for Gene Transfer and Vaccination: Efficient Human Cell Infection and Bypass of Preexisting Adenovirus Immunity

ABSTRACT Replication-deficient human adenovirus type 5 (Ad5) can be produced to high titers in complementing cell lines, such as PER.C6, and is widely used as a vaccine and gene therapy vector. However, preexisting immunity against Ad5 hampers consistency of gene transfer, immunological responses, and vector-mediated toxicities. We report the identification of human Ad35 as a virus with low global prevalence and the generation of an Ad35 vector plasmid system for easy insertion of heterologous genes. In addition, we have identified the minimal sequence of the Ad35-E1B region (molecular weight, 55,000 [55K]), pivotal for complementation of fully E1-lacking Ad35 vector on PER.C6 cells. After stable insertion of the 55K sequence into PER.C6 cells a cell line was obtained (PER.C6/55K) that efficiently transcomplements both Ad5 and Ad35 vectors. We further demonstrate that transduction with Ad35 is not hampered by preexisting Ad5 immunity and that Ad35 efficiently infects dendritic cells, smooth muscle cells, and synoviocytes, in contrast to Ad5.

Pericellular proteases in angiogenesis and vasculogenesis

Pericellular proteases play an important role in angiogenesis and vasculogenesis. They comprise (membrane-type) matrix metalloproteinases [(MT-)MMPs], serine proteases, cysteine cathepsins, and membrane-bound aminopeptidases. Specific inhibitors regulate them. Major roles in initiating angiogenesis have been attributed to MT1-matrix metalloproteinase (MMP), MMP-2, and MMP-9. Whereas MT-MMPs are membrane-bound by nature, MMP-2 and MMP-9 can localize to the membrane by binding to alphavbeta3-integrin and CD44, respectively. Proteases switch on neovascularization by activation, liberation, and modification of angiogenic growth factors and degradation of the endothelial and interstitial matrix. They also modify the properties of angiogenic growth factors and cytokines. Neovascularization requires cell migration, which depends on the assembly of protease-protein complexes at the migrating cell front. MT1-MMP and urokinase (u-PA) form multiprotein complexes in the lamellipodia and focal adhesions of migrating cells, facilitating proteolysis and sufficient support for endothelial cell migration and survival. Excessive proteolysis causes loss of endothelial cell-matrix interaction and impairs angiogenesis. MMP-9 and cathepsin L stimulate the recruitment and action of blood- or bone-marrow-derived accessory cells that enhance angiogenesis. Proteases also generate fragments of extracellular matrix and hemostasis factors that have anti-angiogenic properties. Understanding the complexity of protease activities in angiogenesis contributes to recognizing new targets for stimulation or inhibition of neovascularization in disease.

Wnt but not BMP signaling is involved in the inhibitory action of sclerostin on BMP-stimulated bone formation

Sclerostin is an osteocyte‐derived negative regulator of bone formation. It inhibits BMP‐stimulated bone formation both in vitro and in vivo but has no direct effect on BMP signaling. Instead, sclerostin inhibits Wnt signaling that is required for BMP‐stimulated osteoblastic differentiation.

Toll-Like Receptor 4 Mediates Maladaptive Left Ventricular Remodeling and Impairs Cardiac Function After Myocardial Infarction

Left ventricular (LV) remodeling leads to congestive heart failure and is a main determinant of morbidity and mortality following myocardial infarction. Therapeutic options to prevent LV remodeling are limited, which necessitates the exploration of alternative therapeutic targets. Toll-like receptors (TLRs) serve as pattern recognition receptors within the innate immune system. Activation of TLR4 results in an inflammatory response and is involved in extracellular matrix degradation, both key processes of LV remodeling following myocardial infarction. To establish the role of TLR4 in postinfarct LV remodeling, myocardial infarction was induced in wild-type BALB/c mice and TLR4-defective C3H-Tlr4LPS−d mice. Without affecting infarct size, TLR4 defectiveness reduced the extent of LV remodeling (end-diastolic volume: 103.7±6.8 &mgr;L versus 128.5±5.7 &mgr;L; P<0.01) and preserved systolic function (ejection fraction: 28.2±3.1% versus 16.6±1.3%; P<0.01), as assessed by MRI. In the noninfarcted area, interstitial fibrosis, and myocardial hypertrophy were reduced in C3H-Tlr4LPS−d mice. In the infarcted area, however, collagen density was increased, which was accompanied by fewer macrophages, reduced inflammation regulating cytokine expression levels (interleukin [IL]-1&agr;, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17, tumor necrosis factor-&agr;, interferon-&ggr;, granulocyte/macrophage colony-stimulating factor), and reduced matrix metalloproteinase-2 (4684±515 versus 7573±611; P=0.002) and matrix metalloproteinase-9 activity (76.0±14.3 versus 168.0±36.2; P=0.027). These data provide direct evidence for a causal role of TLR4 in postinfarct maladaptive LV remodeling, probably via inflammatory cytokine production and matrix degradation. TLR4 may therefore constitute a novel target in the treatment of ischemic heart failure.

In Vivo Evidence for a Role of Toll-Like Receptor 4 in the Development of Intimal Lesions

Background—Inflammation plays an important role in atherogenesis. The toll-like receptor 4 (TLR4) is the receptor for bacterial lipopolysaccharides and also recognizes cellular fibronectin and heat shock protein 60, endogenous peptides that are produced in response to tissue injury. To explore a possible role for this receptor in arterial obstructive disease, we determined the expression of TLR4 in the atherosclerotic arterial wall, including adventitia, and studied the effect of adventitial TLR4 activation on neointima formation in a mouse model. Methods and Results—Localization of TLR4 was studied in human atherosclerotic coronary arteries by immunohistochemistry and detected in plaque and adventitia. In the adventitia, not all TLR4-positive cells colocalized with macrophages. In primary human adventitial fibroblasts, expression of TLR4 was demonstrated by immunofluorescence, Western blot, and reverse transcriptase-polymerase chain reaction. Adding lipopolysaccharide to these fibroblasts induced activation of NF-&kgr;B and an increase of mRNAs of various cytokines. The effect of adventitial stimulation of TLR4 was studied in a mouse model. A peri-adventitial cuff was placed around the femoral artery. Application of lipopolysaccharide between cuff and artery augmented neointima formation induced by the cuff (intimal area±SEM, 9134±1714 versus 2353±1076 &mgr;m2, P <0.01). In TLR4-defective mice, application of cuff and lipopolysaccharide resulted in a smaller neointima than in wild-type mice (intimal area, 3859±904 &mgr;m2, P =0.02 versus wild type). Conclusions—A functional TLR4 is expressed in human adventitial fibroblasts and macrophages. Adventitial TLR4 activation augmented neointima formation in a mouse model. These results provide evidence for a link between the immune receptor TLR4 and intimal lesion formation.

Antagomir-mediated silencing of endothelial cell specific microRNA-126 impairs ischemia-induced angiogenesis.

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.

Exploiting the Natural Diversity in Adenovirus Tropism for Therapy and Prevention of Disease

ABSTRACT Since targeting of recombinant adenovirus vectors to defined cell types in vivo is a major challenge in gene therapy and vaccinology, we explored the natural diversity in human adenovirus tissue tropism. Hereto, we constructed a library of Ad5 vectors carrying fibers from other human serotypes. From this library, we identified vectors that efficiently infect human cells that are important for diverse gene therapy approaches and for induction of immunity. For several medical applications (prenatal diagnosis, artificial bone, vaccination, and cardiovascular disease), we demonstrate the applicability of these novel vectors. In addition, screening cell types derived from different species revealed that cellular receptors for human subgroup B adenoviruses are not conserved between rodents and primates. These results provide a rationale for utilizing elements of human adenovirus serotypes to generate chimeric vectors that improve our knowledge concerning adenovirus biology and widen the therapeutic window for vaccination and many different gene transfer applications.

Toll-Like Receptor 4 Is Involved in Outward Arterial Remodeling

Background—Toll-like receptor 4 (Tlr4) is the receptor for exogenous lipopolysaccharides (LPS). Expression of endogenous Tlr4 ligands, heat shock protein 60 (Hsp60) and extra domain A of fibronectin, has been observed in arthritic and oncological specimens in which matrix turnover is an important feature. In atherosclerosis, outward remodeling is characterized by matrix turnover and a structural change in arterial circumference and is associated with a vulnerable plaque phenotype. Since Tlr4 ligands are expressed during matrix turnover, we hypothesized that Tlr4 is involved in arterial remodeling. Methods and Results—In a femoral artery cuff model in the atherosclerotic ApoE3 (Leiden) transgenic mouse, Tlr4 activation by LPS stimulated plaque formation and subsequent outward arterial remodeling. With the use of the same model in wild-type mice, neointima formation and outward remodeling occurred. In Tlr4-deficient mice, however, no outward arterial remodeling was observed independent of neointima formation. Carotid artery ligation in wild-type mice resulted in outward remodeling without neointima formation in the contralateral artery. This was associated with an increase in Tlr4 expression and EDA and Hsp60 mRNA levels. In contrast, outward remodeling was not observed after carotid ligation in Tlr4-deficient mice. Conclusions—These findings provide genetic evidence that Tlr4 is involved in outward arterial remodeling, probably through upregulation of Tlr4 and Tlr4 ligands.

Improved Adenovirus Vectors for Infection of Cardiovascular Tissues

ABSTRACT To identify improved adenovirus vectors for cardiovascular gene therapy, a library of adenovirus vectors based on adenovirus serotype 5 (Ad5) but carrying fiber molecules of other human serotypes, was generated. This library was tested for efficiency of infection of human primary vascular endothelial cells (ECs) and smooth muscle cells (SMCs). Based on luciferase, LacZ, or green fluorescent protein (GFP) marker gene expression, several fiber chimeric vectors were identified that displayed improved infection of these cell types. One of the viruses that performed particularly well is an Ad5 carrying the fiber of Ad16 (Ad5.Fib16), a subgroup B virus. This virus showed, on average, 8- and 64-fold-increased luciferase activities on umbilical vein ECs and SMCs, respectively, compared to the parent vector. GFP andlacZ markers showed that approximately 3-fold (ECs) and 10-fold (SMCs) more cells were transduced. Experiments performed with both cultured SMCs and organ cultures derived from different vascular origins (saphenous vein, iliac artery, left interior mammary artery, and aorta) and from different species demonstrated that Ad5.Fib16 consistently displays improved infection in primates (humans and rhesus monkeys). SMCs of the same vessels of rodents and pigs were less infectable with Ad5.Fib16 than with Ad5. This suggests that either the receptor for human Ad16 is not conserved between different species or that differences in the expression levels of the putative receptor exist. In conclusion, our results show that an Ad5-based virus carrying the fiber of Ad16 is a potent vector for the transduction of primate cardiovascular cells and tissues.

Restenosis after PCI. Part 1: pathophysiology and risk factors

Restenosis is a complex disease for which the pathophysiological mechanisms have not yet been fully elucidated, but are thought to include inflammation, proliferation, and matrix remodeling. Over the years, many predictive clinical, biological, (epi)genetic, lesion-related, and procedural risk factors for restenosis have been identified. These factors are not only useful in risk stratification of patients, they also contribute to our understanding of this condition. Furthermore, these factors provide evidence on which to base treatment tailored to the individual and aid in the development of novel therapeutic modalities. In this Review, we will evaluate the available evidence on the pathophysiological mechanisms of restenosis and provide an overview of the various risk factors, together with the possible clinical application of this knowledge.