osmotic-drug-delivery-to-ischemic-hindlimbs-perfusion-vasculature

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Cyclooxygenase-1 (COX-1) is the rate-limiting component in the synthesis of prostacyclin (PGI2), an important vasodilator and antithrombotic molecule. In balloon-injured, atherosclerosis-free porcine arteries, COX-1 gene transduction increases PGI2 production, induces durable vasodilation, and reduces thrombus formation. We tested the effectiveness of COX-1 local gene transduction for the prevention of postangioplasty restenosis in atherosclerotic arteries in a hypercholesterolemic rabbit model.

Local gene transduction of cyclooxygenase-1 increases blood flow in injured atherosclerotic rabbit arteries.

The charge state distribution and CID fragmentation of two series of deprotonated oligodeoxynucleotide (ODN) 9-mers (5'-GGTTXTTGG-3' and 5'-CCAAYAACC-3', X/Y = G, C, A, or T) have been studied in detail in an ion trap in an effort to understand the intrinsic properties of DNA in vacuo. The distribution of charge states (-2 to -6) is similar for both the X- and Y-series, with the most abundant being the -4 charge state. The T-rich X-series prefers higher charge states (-6 and -5) than does the Y-series. Calculations show that phosphate groups located nearest a thymine are more acidic than those near an adenine, cytosine, or guanine, thus explaining why the X-series prefers higher charge states. We use the term "charge level" to define the ratio of the charge state to the total number of phosphate groups present in the ODN. We find, consistent with previous studies, that the initial step of fragmentation is loss of nucleobase either as an anion or as a neutral. We observe the former for ODNs with charge levels greater than 50% and the latter for ODNs with charge levels below 50%. The overall anionic base loss follows the trend A(-) > G(-) approximately T(-) > C(-); electrostatic potential calculations indicate that this trend follows delocalization of electron density for each anion, with A(-) being the most stabilized through delocalization. For neutral base loss, thymine (TH) is rarely cleaved, while the preferences for AH, GH, and CH loss vary. Proton affinity (PA) calculations show that a nearby negatively charged phosphate enhances the PA of proximally located nucleobases; this PA enhancement probably plays a role in promoting neutral base loss. The trends differ by charge level. At a charge level of 37.5% (-3 charge state), AH loss is preferred over CH and GH loss, regardless of sequence. However, at a charge level of 25% (-2 charge state), the terminal bases are preferentially lost over the internal bases, regardless of identity. By reconstructing the ODN sequences from structurally informative (a-BH) and w ions, we are able to identify the charge locations for the -3 and -2 charge states. For the -3 charge state, one charge resides on each "most terminal" phosphate, with the third being in the middle. For the -2 charge state, each charge resides on the penultimate phosphate groups. We compare our data to earlier experiments in an effort to generalize trends.

Investigation of the initial fragmentation of oligodeoxynucleotides in a quadrupole ion trap: charge level-related base loss.

We report herein a systematic mass spectrometric study of a series of thirty-one non-self-complementary, matched, DNA duplexes ranging in size from 5- to 12-mers. The purpose of this work is threefold: (1) to establish the viability of using mass spectrometry as a tool for examining solution phase stabilities of DNA duplexes; (2) to systematically assess gas-phase stabilities of DNA duplexes; and (3) to compare gas and solution phase stabilities in an effort to understand how media affects DNA stability. These fundamental issues are of importance both on their own, and also for harnessing the potential of mass spectrometry for biological applications. We have found that ion abundances do not always track with solution phase stability; GC content must be taken into account. Two duplexes with the same Tm yet with differing GC content can yield different ion abundances. That is, if two duplexes have the exact same melting temperature, yet one has a higher GC content, the duplex with the higher GC content yields a higher ion abundance. It thus appears that not only is a GC base pair stronger than an AT base pair, but the relative strengths of each differ in the gas phase versus in solution, such that the electrospray process can differentiate between them. We also characterize the gas-phase stabilities of the duplexes, using collision-induced dissociation (CID) as a method to assess stability. We focus on two aspects of this CID experiment. One, we examine what factors appear to control whether the duplexes dissociate into single strands or covalently fragment; we are able to utilize a charge state normalization we coin "charge level" to compare our results with others' and establish generalities regarding dissociation versus fragmentation patterns. Two, we examine those duplexes that primarily dissociate and use CID to assess the gas-phase stabilities. We find that correlation of gas-phase to solution-phase stabilities is more likely to occur when duplexes of varying GC content are examined. Duplexes with the same GC content tend to have stabilities that do not parallel those in solution. We discuss these results in light of the different roles that hydrogen bonding and base stacking play in solution versus the gas phase. Ultimately, we apply what we learn to lend insight into the biological problem of how the carcinogenic, damaged nucleobase O6-methylguanine causes mutations.

DNA stability in the gas versus solution phases: a systematic study of thirty-one duplexes with varying length, sequence, and charge level.

A new class of water-soluble C60 transfecting agents has been prepared using Hirsch-Bingel chemistry and assessed for their ability to act as gene-delivery vectors in vitro. In an effort to elucidate the relationship between the hydrophobicity of the fullerene core, the hydrophilicity of the water-solubilizing groups, and the overall charge state of the C60 vectors in gene delivery and expression, several different C60 derivatives were synthesized to yield either positively charged, negatively charged, or neutral chemical functionalities under physiological conditions. These fullerene derivatives were then tested for their ability to transfect cells grown in culture with DNA carrying the green fluorescent protein (GFP) reporter gene. Statistically significant expression of GFP was observed for all forms of the C60 derivatives when used as DNA vectors and compared to the ability of naked DNA alone to transfect cells. However, efficient in vitro transfection was only achieved with the two positively charged C60 derivatives, namely, an octa-amino derivatized C60 and a dodeca-amino derivatized C60 vector. All C60 vectors showed an increase in toxicity in a dose-dependent manner. Increased levels of cellular toxicity were observed for positively charged C60 vectors relative to the negatively charged and neutral vectors. Structural analyses using dynamic light scattering and optical microscopy offered further insights into possible correlations between the various derivatized C60 compounds, the C60 vector/DNA complexes, their physical attributes (aggregation, charge) and their transfection efficiencies. Recently, similar Gd@C60-based compounds have demonstrated potential as advanced contrast agents for magnetic resonance imaging (MRI). Thus, the successful demonstration of intracellular DNA uptake, intracellular transport, and gene expression from DNA using C60 vectors suggests the possibility of developing analogous Gd@C60-based vectors to serve simultaneously as both therapeutic and diagnostic agents.

Water-soluble fullerene (C60) derivatives as nonviral gene-delivery vectors.

Adequate expression of surfactant protein-B (SP-B) is critical in the function of pulmonary surfactant to reduce alveolar surface tension. Expression of SP-B mRNA is restricted to specific lung-airway epithelial cells, and human SP-B mRNA stability is increased in the presence of the synthetic glucocorticoid dexamethasone (DEX). Although the mechanism of SP-B mRNA stabilization by DEX is unknown, studies suggest involvement of the glucocorticoid receptor (GR). We developed a dual-cistronic plasmid-based expression assay in which steady-state levels of SP-B mRNA, determined by Northern analysis, reproducibly reflect changes in SP-B mRNA stability. Using this assay, we found that steady-state levels of SP-B mRNA increased greater than twofold in transfected human-airway epithelial cells (A549) incubated with DEX (10(-7) M). DEX-mediated changes in SP-B mRNA levels required the presence of the SP-B mRNA 3'-untranslated region but did not require ongoing protein synthesis. The effect of DEX on SP-B mRNA levels was dose dependent, with maximal effect at 10(-7) M. DEX increased levels of SP-B mRNA in cells lacking GR, and the presence of the GR antagonist RU486 did not interfere with the effect of DEX. Surprisingly, other steroid hormones (progesterone, estradiol, and vitamin D; 10(-7) M) significantly increased SP-B mRNA levels, suggesting a common pathway of steroid hormone action on SP-B mRNA stability. These results indicate that the effect of DEX to increase SP-B mRNA stability is independent of activated GR and suggests that the mechanism is mediated by posttranscriptional or nongenomic effects of glucocorticoids.

Glucocorticoid regulation of human pulmonary surfactant protein-B (SP-B) mRNA stability is independent of activated glucocorticoid receptor.

BACKGROUND: To examine prevalence and associated factors of diabetic retinopathy in patients with Type 2 diabetes mellitus in urban communities of Beijing. METHODS: The community health care center-based study included subjects with diabetes mellitus and an age of 20 years to 80 years, who were recruited from 15 community health centers in urban Beijing. Diabetes mellitus was defined using the World Health Organization criteria. Fundus photographs were graded using the modified Airlie House classification system. RESULTS: Of 2,642 eligible patients, 2,007 (76.0%) subjects (1,199 women) with a mean age of 64.1 Â± 9.0 years participated. The overall prevalence of diabetic retinopathy was 24.7 Â± 1.0% (95% confidence interval [CI], 22.8-26.6). In binary logistic analysis, presence of diabetic retinopathy was associated with younger age (odds ratio [OR], 0.97; 95% CI, 0.95-0.98), longer duration of diabetes (OR, 1.10; 95% CI, 1.08-1.12), higher concentration of glycosylated hemoglobin HbA1c (OR, 1.23; 95% CI, 1.14-1.33), higher systolic blood pressure (OR, 1.01; 95% CI, 1.01-1.02), lower body mass index (OR, 0.95; 95% CI, 0.92-0.98), and elevated blood urea concentration (OR, 1.01; 95% CI, 1.00-1.01). Microalbuminuria was an additional associated factor (OR, 1.55; 95% CI, 1.16-2.08). Patients with microalbuminuria were 4.7 times more likely to have a severe or proliferating diabetic retinopathy than those without microalbuminuria. CONCLUSION: In the urban population of Beijing, prevalence of diabetic retinopathy in diabetic patients was 25%. As in whites, increased blood pressure besides elevated plasma glucose concentrations was highly significantly associated with diabetic retinopathy in Chinese. It suggests that in Chinese as also in whites, blood pressure control beside control of plasma glucose levels is important to prevent development or progression of diabetic retinopathy.

PREVALENCE AND RISK FACTORS FOR DIABETIC RETINOPATHY: The Beijing Communities Diabetes Study 6.

Hintergrund: um zu pr&uuml;fen geben Sie Pr&auml;valenz und Umfeldfaktoren der diabetischen Retinopathie bei Patienten mit 2 Diabetes Mellitus in st&auml;dtischen Gemeinden von Peking. Methoden: Die Gemeinschaft Gesundheits Zentrum-basierte Studie umfasste Probanden mit Diabetes Mellitus und ein Alter von 20 Jahren bis 80 Jahren, die aus 15 Gemeinschaft Gesundheitszentren in st&auml;dtischen Peking rekrutiert wurden. Diabetes Mellitus wurde anhand der Kriterien der Weltgesundheitsorganisation (WHO) definiert. IOL-Fotografien wurden mit Hilfe des modifizierten Airlie House-Klassensystems benotet. Ergebnisse: Von 2.642 Patienten nahmen 2.007 (76,0 %) Themen (1.199 Frauen) mit einem Durchschnittsalter von 64.1 &plusmn; 9,0 Jahren. Die Gesamtpr&auml;valenz der diabetischen Retinopathie war 24,7 &plusmn; 1,0 % (95 % Konfidenzintervall [CI], 22,8-26.6). In bin&auml;re logistische Analyse wurde Pr&auml;senz der diabetischen Retinopathie mit j&uuml;ngerem Alter (Odds Ratio [OR], 0.97; 95 % CI, 0.95-0.98), l&auml;ngere Dauer des Diabetes (OR, 1.10; 95 % CI, 1.08-1.12), h&ouml;here Konzentration von glykosiliertes H&auml;moglobin HbA1c (OR, 1.23; 95 % CI, 1.14-1,33), h&ouml;here systolische Blutdruck (OR, 1.01; 95 % CI, 1.01-1.02), niedriger Body-mass-Index (OR, 0.95; 95 % CI, 0.92-0.98), und erh&ouml;hte Konzentration im Blut Harnstoff (OR1.01; 95 % CI, 1.00-1.01). Mikroalbuminurie wurde ein zus&auml;tzlicher zugeordneten Faktor (OR, 1,55; 95 % CI, 1.16-2.08). Patienten mit Mikroalbuminurie waren 4,7 Mal h&auml;ufiger an einer schweren oder proliferativen Diabetische Retinopathie als solche ohne Mikroalbuminurie zu haben. Fazit: In der st&auml;dtischen Bev&ouml;lkerung von Peking war die Pr&auml;valenz der diabetischen Retinopathie bei diabetischen Patienten 25 %. Wie in wei&szlig;en wurde erh&ouml;hter Blutdruck neben erh&ouml;hten Plasma-Glukose-Konzentrationen sehr deutlich mit Diabetische Retinopathie in Chinesisch. Er schl&auml;gt, die in der chinesischen als auch in wei&szlig;en, Blutdruck Kontrolle neben Kontrolle der Glukose-Plasmaspiegel wichtig ist, die Entwicklung oder Progression der diabetischen Retinopathie zu verhindern.

Pr&auml;valenz und Risikofaktoren f&uuml;r Diabetische RETINOPATHIE: Beijing Gemeinschaften Diabetes Studie 6.

The ability of pulmonary surfactant to reduce alveolar surface tension requires adequate expression of surfactant protein B (SP-B). Dexamethasone (DEX, 10(-7) M) increases human SP-B mRNA stability by a mechanism that requires a 126-nt-long segment (the 7.6S region) of the 3'-untranslated region (3'-UTR). The objective of this study was to identify sequences in the 7.6S region that mediate regulation of SP-B mRNA stability. The 7.6S region was found to be sufficient for DEX-mediated stabilization of mRNA. Sequential substitution mutagenesis of the 7.6S region indicates that a 90-nt region is required for DEX-mediated stabilization and maintenance of intrinsic stability. In this region, one 30-nt-long element (002), predicted to form a stem-loop structure, is sufficient for DEX-mediated stabilization of mRNA and intrinsic mRNA stability. Cytosolic proteins specifically bind element 002, and binding activity is unaffected whether proteins are isolated from cells incubated in the absence or presence of DEX. While loop sequences of element 002 have no role in regulation of SP-B mRNA stability, the proximal stem sequences are required for DEX-mediated stabilization and specific binding of proteins. Mutation of the sequences that comprise the proximal or distal arm of the stem negates the destabilizing activity of element 002 on intrinsic SP-B mRNA stability. These results indicate that cytosolic proteins bind a single hairpin structure that mediates intrinsic and hormonal regulation of SP-B mRNA stability via mechanisms that involve sequences of the stems of the hairpin structure.

Sequences of a hairpin structure in the 3'-untranslated region mediate regulation of human pulmonary surfactant protein B mRNA stability.

Prostacyclin and its stable analogs play an important vascular protective role by promoting angiogenesis, but their role in arteriolar growth is unclear. Here, we examined the effect of prostacyclin stable analog carbaprostacyclin on arteriolar growth in mouse hindlimb ischemia. Using an osmotic-controlled release system to continuously deliver carbaprostacyclin or saline (control) to ischemic mouse hindlimbs for up to 14 days, we found that blood perfusion was significantly better at 7 and 14 days in carbaprostacyclin-treated mice than in saline-treated mice. Microscopic examination of the microvasculature showed more morphological signs of arteriolar formation in carbaprostacyclin- versus saline-treated legs. A double-blind, quantitative microcomputed tomography analysis indicated that carbaprostacyclin-treated legs had markedly increased vascular volume and small- to medium-sized vessel numbers that correspond to decreased vessel separation. A proteome profiler antibody array demonstrated that carbaprostacyclin-treated ischemic muscles secreted significantly higher amounts of acidic fibroblast growth factor and other chemokines. Conditioned media containing those secreted factors promoted smooth muscle cell growth and migration. Additionally, increased acidic fibroblast growth factor protein levels were detected in smooth muscle cells and skeletal myotubes at different time periods after carbaprostacyclin treatment. Furthermore, the selective peroxisome proliferation-activated receptor Î²/Î´ antagonist significantly suppressed carbaprostacyclin-induced acidic fibroblast growth factor protein production. Collectively, our data provide the first morphological and molecular evidence that local delivery of carbaprostacyclin promotes vascular growth in hindlimb ischemia, and that peroxisome proliferation-activated receptor Î²/Î´ signaling plays a critical role in inducing acidic fibroblast growth factor expression.

Article	Year
Local gene transduction of cyclooxygenase-1 increases blood flow in injured atherosclerotic rabbit arteries. Circulation\| PubMed ID: 15824211	2005
Investigation of the initial fragmentation of oligodeoxynucleotides in a quadrupole ion trap: charge level-related base loss. Journal of the American Society for Mass Spectrometry\| PubMed ID: 16198120	2005
DNA stability in the gas versus solution phases: a systematic study of thirty-one duplexes with varying length, sequence, and charge level. Journal of the American Society for Mass Spectrometry\| PubMed ID: 16914323	2006
Water-soluble fullerene (C60) derivatives as nonviral gene-delivery vectors. Molecular pharmaceutics\| PubMed ID: 18505267	2008
Glucocorticoid regulation of human pulmonary surfactant protein-B (SP-B) mRNA stability is independent of activated glucocorticoid receptor. American journal of physiology. Lung cellular and molecular physiology\| PubMed ID: 21398497	2011
PREVALENCE AND RISK FACTORS FOR DIABETIC RETINOPATHY: The Beijing Communities Diabetes Study 6. Retina (Philadelphia, Pa.)\| PubMed ID: 21886023	2012
Sequences of a hairpin structure in the 3'-untranslated region mediate regulation of human pulmonary surfactant protein B mRNA stability. American journal of physiology. Lung cellular and molecular physiology\| PubMed ID: 22367784	2012
Targeted delivery of carbaprostacyclin to ischemic hindlimbs enhances adaptive remodeling of the microvascular network. Hypertension\| PubMed ID: 23529172	2013

Su Pan

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