Yanto Ridwan

Nucleotide Excision DNA Repair Is Associated With Age-Related Vascular Dysfunction

Background Vascular dysfunction in atherosclerosis and diabetes mellitus, as observed in the aging population of developed societies, is associated with vascular DNA damage and cell senescence. We hypothesized that cumulative DNA damage during aging contributes to vascular dysfunction. Methods and Results In mice with genomic instability resulting from the defective nucleotide excision repair genes ERCC1 and XPD (Ercc1d/− and XpdTTD mice), we explored age-dependent vascular function compared with that in wild-type mice. Ercc1d/− mice showed increased vascular cell senescence, accelerated development of vasodilator dysfunction, increased vascular stiffness, and elevated blood pressure at a very young age. The vasodilator dysfunction was due to decreased endothelial nitric oxide synthase levels and impaired smooth muscle cell function, which involved phosphodiesterase activity. Similar to Ercc1d/− mice, age-related endothelium-dependent vasodilator dysfunction in XpdTTD animals was increased. To investigate the implications for human vascular disease, we explored associations between single-nucleotide polymorphisms of selected nucleotide excision repair genes and arterial stiffness within the AortaGen Consortium and found a significant association of a single-nucleotide polymorphism (rs2029298) in the putative promoter region of DDB2 gene with carotid-femoral pulse wave velocity. Conclusions Mice with genomic instability recapitulate age-dependent vascular dysfunction as observed in animal models and in humans but with an accelerated progression compared with wild-type mice. In addition, we found associations between variations in human DNA repair genes and markers for vascular stiffness, which is associated with aging. Our study supports the concept that genomic instability contributes importantly to the development of cardiovascular disease.

Synaptic proteome changes in a DNA repair deficient ercc1 mouse model of accelerated aging

Cognitive decline is one of the earliest hallmarks of both normal and pathological brain aging. Here we used Ercc1 mutant mice, which are impaired in multiple DNA repair systems and consequently show accelerated aging and progressive memory deficits, to identify changes in the levels of hippocampal synaptic proteins that potentially underlie these age-dependent deficits. Aged Ercc1 mutant mice show normal gross hippocampal dendritic morphology and synapse numbers, and Ercc1 mutant hippocampal neurons displayed normal outgrowth and synapse formation in vitro. However, using isobaric tag for relative and absolute quantification (iTRAQ) of hippocampal synaptic proteins at two different ages, postnatal days 28 and 112, we observed a progressive decrease in synaptic ionotropic glutamate receptor levels and increased levels of G-proteins and of cell adhesion proteins. These together may cause long-term changes in synapse function. In addition, we observed a downregulation of mitochondrial proteins and concomitant upregulation of Na,K-ATPase subunits, which might compensate for reduced mitochondrial activity. Thus, our findings show that under conditions of apparent intact neuronal connectivity, levels of specific synaptic proteins are already affected during the early stages of DNA damage-induced aging, which might contribute to age-dependent cognitive decline.

Accelerated loss of hearing and vision in the DNA-repair deficient Ercc1δ/− mouse

Age-related loss of hearing and vision are two very common disabling conditions, but the underlying mechanisms are still poorly understood. Damage by reactive oxygen species and other reactive cellular metabolites, which in turn may damage macromolecules such as DNA, has been implicated in both processes. To investigate whether DNA damage can contribute to age-related hearing and vision loss, we investigated hearing and vision in Ercc1(δ/-) mutant mice, which are deficient in DNA repair of helix-distorting DNA lesions and interstrand DNA crosslinks. Ercc1(δ/-) mice showed a progressive, accelerated increase of hearing level thresholds over time, most likely arising from deteriorating cochlear function. Ercc1(δ/-) mutants also displayed a progressive decrease in contrast sensitivity followed by thinning of the outer nuclear layer of the eyeball. The strong parallels with normal ageing suggest that unrepaired DNA damage can induce age-related decline of the auditory and visual system.

Timelines of the “free-particle” and “fixed-particle” models of stone-formation: theoretical and experimental investigations

Two major theories on renal stone formation will be reviewed, the “free-particle” and “fixed-particle” mechanisms. These theories combine data on intrinsic factors (inborn metabolic errors), extrinsic factors (diet), renal cell responses and the physico-chemistry and biochemistry of urine into mechanisms of stone formation. This paper describes the specific role of time in both mechanisms. The timeline of crystal- and stone formation was deducted from literature data and was measured for two stones using radioisotope decay analysis. The stones of similar size and composition showed, respectively, a timeline of a few years and a development that took decades. In combination with data on stone architecture and patient characteristics these timelines are explained using the free-particle and fixed-particle mechanisms. Consideration of the timeline of stone formation has clinical implications. We conclude that the fixed-particle mechanism can be a slow process where decades pass between the first formation of a precipitate in the renal interstitium and the clinical presentation of the stone. Added to the fact that the mechanism of this initial precipitation is still ill defined, the conditions that started fixed-particle stone formation in an individual patient can be obscure. Blood and urine analysis in such patients does not necessarily reveal the individual’s risk for recurrence as lifestyle may have changed over time. This is in fact what defines the so-called idiopathic stoneformers. For these patients, prevention of outgrowth of previously formed precipitates, papillary plaques, may be more relevant than prevention of new plaque formation. In contrast, a patient who has formed a stone in a relatively short time through the free-particle mechanism is more likely to show abnormal values in blood and urine that explain the starting event of stone formation. In these patients, measurement of such values provides useful information to guide preventive measures.

Evaluation of a Fluorescent and Radiolabeled Hybrid Somatostatin Analog In Vitro and in Mice Bearing H69 Neuroendocrine Xenografts

In the treatment of neuroendocrine tumors (NETs), complete surgical removal of malignancy is generally desirable, because it offers curative results. Preoperative guidance with radiolabeled somatostatin analogs, commonly used for NET diagnosis and preoperative planning, is limited by its low resolution, with the risk that tumor margins and small metastases will be incompletely resected with subsequent recurrence. A single hybrid probe combining radiotracer and optical dye would enable high-resolution optical guidance, also during surgery. In the current study, the hybrid labeled somatostatin analog Cy5-DTPA-Tyr3-octreotate (DTPA is diethylene triamine pentaacetic acid) was synthesized and evaluated for its ability to specifically trace NET cells in vitro and in an animal model. The performance of the hybrid tracer was compared with that of octreotate with only radiolabel or only optical label. Methods: The binding affinity and internalization capacity of Cy5-DTPA-Tyr3-octreotate were assessed in vitro. Biodistribution profiles and both nuclear and optical in vivo imaging of Cy5-111In -DTPA-Tyr3-octreotate were performed in NET-bearing mice and compared with the performance of 111In-DTPA-Tyr3-octreotate. Results: In vitro studies showed a low receptor affinity and internalization rate for Cy5-DTPA-Tyr3-octreotate. The dissociation constant value was 387.7 ± 97.9 nM for Cy5-DTPA-Tyr3-octreotate, whereas it was 120.5 ± 18.1 nM for DTPA-Tyr3-octreotate. Similarly, receptor-mediated internalization reduced from 33.76% ± 1.22% applied dose for DTPA-Tyr3-octreotate to 1.32% ± 0.02% applied dose for Cy5-DTPA-Tyr3-octreotate. In contrast, in vivo and ex vivo studies revealed similar tumor uptake values of Cy5-111In-DTPA-Tyr3-octreotate and 111In -DTPA-Tyr3-octreotate (6.93 ± 2.08 and 5.16 ± 1.27, respectively). All organs except the kidneys showed low background radioactivity, with especially low activities in the liver, and high tumor-to-tissue ratios were achieved—both favorable for the tracer’s toxicity profile. Hybrid imaging in mice confirmed that the nuclear and fluorescence signals colocalized. Conclusion: The correlation between findings with the optical and the nuclear probes underlines the potential of combining SPECT imaging with fluorescence guidance and shows the promise of this novel hybrid peptide for preoperative and intraoperative imaging of NET.

AT1-receptor blockade, but not renin inhibition, reduces aneurysm growth and cardiac failure in fibulin-4 mice

Aims: Increasing evidence supports a role for the angiotensin II-AT1-receptor axis in aneurysm development. Here, we studied whether counteracting this axis via stimulation of AT2 receptors is beneficial. Such stimulation occurs naturally during AT1-receptor blockade with losartan, but not during renin inhibition with aliskiren. Methods and results: Aneurysmal homozygous fibulin-4R/R mice, displaying a four-fold reduced fibulin-4 expression, were treated with placebo, losartan, aliskiren, or the &bgr;-blocker propranolol from day 35 to 100. Their phenotype includes cystic media degeneration, aortic regurgitation, left ventricular dilation, reduced ejection fraction, and fractional shortening. Although losartan and aliskiren reduced hemodynamic stress and increased renin similarly, only losartan increased survival. Propranolol had no effect. No drug rescued elastic fiber fragmentation in established aneurysms, although losartan did reduce aneurysm size. Losartan also increased ejection fraction, decreased LV diameter, and reduced cardiac pSmad2 signaling. None of these effects were seen with aliskiren or propranolol. Longitudinal micro-CT measurements, a novel method in which each mouse serves as its own control, revealed that losartan reduced LV growth more than aneurysm growth, presumably because the heart profits both from the local (cardiac) effects of losartan and its effects on aortic root remodeling. Conclusion: Losartan, but not aliskiren or propranolol, improved survival in fibulin-4R/R mice. This most likely relates to its capacity to improve structure and function of both aorta and heart. The absence of this effect during aliskiren treatment, despite a similar degree of blood pressure reduction and renin–angiotensin system blockade, suggests that it might be because of AT2-receptor stimulation.

Aortic microcalcification is associated with elastin fragmentation in Marfan syndrome

Marfan syndrome (MFS) is a connective tissue disorder in which aortic rupture is the major cause of death. MFS patients with an aortic diameter below the advised limit for prophylactic surgery (<5 cm) may unexpectedly experience an aortic dissection or rupture, despite yearly monitoring. Hence, there is a clear need for improved prognostic markers to predict such aortic events. We hypothesize that elastin fragments play a causal role in aortic calcification in MFS, and that microcalcification serves as a marker for aortic disease severity. To address this hypothesis, we analysed MFS patient and mouse aortas. MFS patient aortic tissue showed enhanced microcalcification in areas with extensive elastic lamina fragmentation in the media. A causal relationship between medial injury and microcalcification was revealed by studies in vascular smooth muscle cells (SMCs); elastin peptides were shown to increase the activity of the calcification marker alkaline phosphatase (ALP) and reduce the expression of the calcification inhibitor matrix GLA protein in human SMCs. In murine Fbn1C1039G/+ MFS aortic SMCs, Alpl mRNA and activity were upregulated as compared with wild‐type SMCs. The elastin peptide‐induced ALP activity was prevented by incubation with lactose or a neuraminidase inhibitor, which inhibit the elastin receptor complex, and a mitogen‐activated protein kinase kinase‐1/2 inhibitor, indicating downstream involvement of extracellular signal‐regulated kinase‐1/2 (ERK1/2) phosphorylation. Histological analyses in MFS mice revealed macrocalcification in the aortic root, whereas the ascending aorta contained microcalcification, as identified with the near‐infrared fluorescent bisphosphonate probe OsteoSense‐800. Significantly, microcalcification correlated strongly with aortic diameter, distensibility, elastin breaks, and phosphorylated ERK1/2. In conclusion, microcalcification co‐localizes with aortic elastin degradation in MFS aortas of humans and mice, where elastin‐derived peptides induce a calcification process in SMCs via the elastin receptor complex and ERK1/2 activation. We propose microcalcification as a novel imaging marker to monitor local elastin degradation and thus predict aortic events in MFS patients. Copyright

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

Optical Imaging of Tumor Response to Hyperbaric Oxygen Treatment and Irradiation in an Orthotopic Mouse Model of Head and Neck Squamous Cell Carcinoma

PurposeHyperbaric oxygen therapy (HBOT) is used in the treatment of radiation-induced tissue injury but its effect on (residual) tumor tissue is indistinct and therefore investigated in this study.ProceduresOrthotopic FaDu tumors were established in mice, and the response of the (irradiated) tumors to HBOT was monitored by bioluminescence imaging. Near infrared fluorescence imaging using AngioSense750 and Hypoxisense680 was applied to detect tumor vascular permeability and hypoxia.ResultsHBOT treatment resulted in accelerated growth of non-irradiated tumors, but mouse survival was improved. Tumor vascular leakiness and hypoxia were enhanced after HBOT, whereas histological characteristics, epithelial-to-mesenchymal transition markers, and metastatic incidence were not influenced.ConclusionsSquamous cell carcinoma responds to HBOT with respect to tumor growth, vascular permeability, and hypoxia, which may have implications for its use in cancer patients. The ability to longitudinally analyze tumor characteristics highlights the versatility and potential of optical imaging methods in oncological research.

A multimodal imaging approach based on micro-CT and fluorescence molecular tomography for longitudinal assessment of bleomycin-induced lung fibrosis in mice

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which causes significant deterioration in lung function and subsequent death from respiratory failure. The pathogenesis of IPF in experimental animal models has been induced by bleomycin administration. In this study, we investigate an IPF-like mouse model induced by a double intratracheal bleomycin instillation. Standard histological assessments used for studying lung fibrosis are invasive terminal procedures. The goal of this work is to monitor lung fibrosis through noninvasive imaging techniques such as Fluorescent Molecular Tomography (FMT) and Micro-CT. These two technologies validated with histology findings could represent a revolutionary functional approach for real time non-invasive monitoring of IPF disease severity and progression. The fusion of different approaches represents a step further for understanding the IPF disease, where the molecular events occurring in a pathological condition can be observed with FMT and the subsequent anatomical changes can be monitored by Micro-CT.