Abstract 249: Delayed Gene Transfer Increases Transgene Expression in Vein Grafts

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Liang Du ◽  
Jingwan Zhang ◽  
Alexander Clowes ◽  
David Dichek
Keyword(s):  
Gene Expression ◽  
Blood Flow ◽  
Gene Transfer ◽  
Transgene Expression ◽  
Ex Vivo ◽  
Arterial Blood Flow ◽  
Vein Grafts ◽  
Arterial Blood ◽  
En Face

Background Autogenous vein grafts are effective therapies for obstructive arterial disease. However, their long-term utility is limited by stenosis and occlusion. Genetic engineering of veins that prevents intimal hyperplasia and atherosclerosis could significantly improve the clinical utility of vein grafts. We recently reported that a helper-dependent adenoviral vector (HDAd) reduces atherosclerosis 4 wks after gene transfer in fat-fed rabbits and can express a therapeutic transgene (apo AI) in normal rabbit carotids for at least 48 wks. Use of HDAd for vein graft gene therapy will depend on achievement of similarly high and persistent transgene expression in grafted veins. Hypothesis We tested the hypothesis that Ad-mediated transgene expression in grafted veins (at an early time point) can be increased by varying the timing of gene transfer. Methods Rabbit external jugular veins were transduced by exposure to a beta galactosidase (b-gal)-expressing Ad: in situ either without (a) or with (b) immediate arterial grafting; c) ex vivo with grafting after overnight incubation with Ad; d) in vivo immediately after grafting and e) in vivo 4 wks after grafting (n = 6 - 19 veins/group). Transgene expression was measured in veins removed 3 d after Ad exposure by PCR quantitation of b-gal mRNA and by en-face planimetry of blue-stained area. Results B-gal transgene expression was higher in ungrafted veins than in veins grafted immediately after gene transfer (84 ± 17 vs 9.4 ± 2.0 arbitrary units (AU); P < 0.0001). Overnight incubation of veins with Ad increased gene expression ex vivo by 10-fold but neither this nor performing vector infusion immediately after grafting improved gene expression (11 ± 4.7 and 9.1 ± 1.8 AU; P > 0.9 for both vs immediately grafted veins). Delaying gene transfer until 4 wks after grafting significantly increased gene expression, to a level equivalent to transgene expression in ungrafted veins (61 ± 11 AU; P = 0.3 vs ungrafted veins). En face planimetry yielded similar results. Conclusions Exposure of a transduced vein to arterial blood flow is associated with significant loss of transgene expression. Transgene expression in grafted veins is significantly higher when gene transfer is performed 4 wks after exposure of the vein to arterial blood flow.

Abstract P308: Rgs2 Promotes Uterine Blood Flow During Pregnancy

Hypertension ◽  
2017 ◽  
Vol 70 (suppl_1) ◽  
Author(s):  
Jennifer N Koch ◽  
Elizabeth A Owens ◽  
Shelby Dahlen ◽  
Jie Li ◽  
Patrick Osei Owusu
Keyword(s):  
Gene Expression ◽  
Blood Pressure ◽  
Blood Flow ◽  
G Protein ◽  
Arterial Blood Flow ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Uterine Blood Flow ◽  
Placental Perfusion ◽  
Arterial Blood

Regulators of G protein signaling (RGS) proteins are crucial in mediating vascular smooth muscle contraction via the regulation of heterotrimeric G proteins, affecting blood pressure and arterial blood flow. Previous studies by others and us showed that RGS2 deficiency augments vascular tone and impairs uterine blood flow (UBF) in non-pregnant mice, and that an Rgs2 loss-of-function mutation is linked to preeclampsia in humans; however, the mechanisms are unclear. Here, we tested the hypothesis that increased RGS2 expression and/or function facilitates placental perfusion by promoting vasodilation and UBF. We determined gene expression throughout pregnancy and post-partum period by real-time qPCR, while uterine blood flow and blood pressure were examined by ultrasound and carotid artery catheterization, respectively, under anesthesia. RGS2 expression decreased markedly by pregnancy day 10 (0.049 ± 0.013 vs. 0.023 ± 0.017) but returned to non-pregnancy level by day 15 (0.049 ± 0.013 vs. 0.041 ± 0.008,) in wild type mice. The pattern of changes in impedance to UBF mimicked gene expression profile in WT mice; in contrast, impedance remained elevated in Rgs2-/- mice at pregnancy day 15 (RI; WT: 0.516 ± 0.027, vs. RGS2-/-: 0.714 ± 0.020). Systemic blood pressure was similar between WT and Rgst2-/- mice at all stages of pregnancy. The results together indicate that RGS2 promotes uterine perfusion during pregnancy independently of its blood pressure effects. These findings are clinically relevant as selective targeting of G protein signaling could improve utero-placental hypoperfusion during pregnancy and prevent the development of pregnancy complications such as preeclampsia.

Spiral Laminar Flow in Vivo

1996 ◽  
Vol 91 (1) ◽  
pp. 17-21 ◽  
Author(s):  
P. A. Stonebridge ◽  
P. R. Hoskins ◽  
P.L. Allan ◽  
J. F. F. Belch
Keyword(s):  
Blood Flow ◽  
Reverse Flow ◽  
Arterial Disease ◽  
Flow Patterns ◽  
Endothelial Damage ◽  
Arterial Blood Flow ◽  
Arterial Tree ◽  
Arterial Blood ◽  
Blood Flow Patterns

1. Blood flow patterns are poorly understood despite their impact on arterial disease. There have been few measurements in vivo of the three-dimensional blood flow patterns; we present the results of such studies using a new non-invasive in-vivo method of examining biplanar arterial blood flow patterns. 2. Multiple colour Doppler ultrasound directional velocity images were obtained at two different beam target angles from the artery in the plane perpendicular to its axis. Ensemble average images were constructed; the absolute velocity and direction were calculated by compounding the left and right averaged images. Simple directional, non-directional velocity and vector maps were constructed. 3. Flow patterns were sampled in 11 healthy male volunteers at four points of the pulse cycle; peak systole, systolic downswing, diastolic reverse flow and diastolic forward flow and at three sites; the right common and distal superficial femoral and the left common femoral arteries. 4. Stable rotational flow was observed in all subjects, the direction of rotation varying between sides and individuals. 5. There are theoretical advantages to spiral laminar blood flow; the forward-directed, rotationally induced stability and reduction of laterally directed forces may reduce turbulence in the tapering branching arterial tree and at stenoses and have a beneficial effect on mechanisms of endothelial damage and repair.

scholarly journals In vivo gene transfer into rat bone marrow progenitor cells using rSV40 viral vectors

Blood ◽  
2005 ◽  
Vol 106 (8) ◽  
pp. 2655-2662 ◽  
Author(s):  
Bianling Liu ◽  
Judy Daviau ◽  
Carmen N. Nichols ◽  
David S. Strayer
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Transgene Expression ◽  
Viral Vectors ◽  
Ex Vivo ◽  
Simian Virus 40 ◽  
Hematopoietic Stem ◽  
Entire Study

AbstractHematopoietic stem cell (HSC) gene transfer has been attempted almost entirely ex vivo and has been limited by cytokine-induced loss of self-renewal capacity and transplantation-related defects in homing and engraftment. Here, we attempted to circumvent such limitations by injecting vectors directly into the bone marrow (BM) to transduce HSCs in their native environment. Simian virus 40 (SV40)–derived gene delivery vectors were used because they transduce resting CD34+ cells very efficiently. Rats received SV-(Nef-FLAG), carrying FLAG marker epitope—or a control recombinant SV40 (rSV40)—directly into both femoral marrow cavities. Intracellular transgene expression by peripheral blood (PB) or BM cells was detected by cytofluorimetry. An average of 5.3% PB leukocytes expressed FLAG for the entire study—56 weeks. Transgene expression was sustained in multiple cell lineages, including granulocytes (average, 3.3% of leukocytes, 20.4% of granulocytes), CD3+ T lymphocytes (average, 0.53% of leukocytes, 1% of total T cells), and CD45R+ B lymphocytes, indicating gene transfer to long-lived progenitor cells with multilineage capacity. An average of 15% of femoral marrow cells expressed FLAG up to 16.5 months after transduction. Thus, direct intramarrow administration of rSV40s yields efficient gene transfer to rat BM progenitor cells and may be worthy of further investigation.

scholarly journals Endothelial miR-17∼92 cluster negatively regulates arteriogenesis via miRNA-19 repression of WNT signaling

2015 ◽  
Vol 112 (41) ◽  
pp. 12812-12817 ◽  
Author(s):  
Shira Landskroner-Eiger ◽  
Cong Qiu ◽  
Paola Perrotta ◽  
Mauro Siragusa ◽  
Monica Y. Lee ◽  
...  
Keyword(s):  
Blood Flow ◽  
Wnt Signaling ◽  
Critical Role ◽  
Arterial Blood Flow ◽  
In Vivo Model ◽  
Mirna Regulation ◽  
Arterial Blood ◽  
Specific Deletion

The contribution of endothelial-derived miR-17∼92 to ischemia-induced arteriogenesis has not been investigated in an in vivo model. In the present study, we demonstrate a critical role for the endothelial-derived miR-17∼92 cluster in shaping physiological and ischemia-triggered arteriogenesis. Endothelial-specific deletion of miR-17∼92 results in an increase in collateral density limbs and hearts and in ischemic limbs compared with control mice, and consequently improves blood flow recovery. Individual cluster components positively or negatively regulate endothelial cell (EC) functions in vitro, and, remarkably, ECs lacking the cluster spontaneously form cords in a manner rescued by miR-17a, -18a, and -19a. Using both in vitro and in vivo analyses, we identified FZD4 and LRP6 as targets of miR-19a/b. Both of these targets were up-regulated in 17∼92 KO ECs compared with control ECs, and both were shown to be targeted by miR-19 using luciferase assays. We demonstrate that miR-19a negatively regulates FZD4, its coreceptor LRP6, and WNT signaling, and that antagonism of miR-19a/b in aged mice improves blood flow recovery after ischemia and reduces repression of these targets. Collectively, these data provide insights into miRNA regulation of arterialization and highlight the importance of vascular WNT signaling in maintaining arterial blood flow.

Pre-clinical pharmacology of zolmitriptan (Zomig™; formerly 311C90), a centrally and peripherally acting 5HT1B/1D agonist for migraine

Cephalalgia ◽  
1997 ◽  
Vol 17 (18_suppl) ◽  
pp. 4-14 ◽  
Author(s):  
Gr Martin
Keyword(s):  
Blood Flow ◽  
Nucleus Tractus Solitarius ◽  
Ex Vivo ◽  
Partial Agonist ◽  
Venous Blood ◽  
Arterial Blood Flow ◽  
Vascular Effects ◽  
Protein Extravasation ◽  
Trigeminal Stimulation ◽  
Arterial Blood

Zolmitriptan (Zomig™ formerly 311C90) is a novel 5-hydroxytryptamine (5HT)1B/1D receptor agonist with proven efficacy in the acute treatment of migraine with or without preceding aura. The drug differs from presently available members of this drug class in that it combines 5HT1B/1D receptor partial agonist activity with robust oral pharmacokinetics and an ability to inhibit trigeminovascular activation centrally as well as peripherally in preclinical studies. Consistent with its selectivity for 5HT1B/1D receptors, zolmitriptan produces constriction of various isolated blood vessels, most notably cranial arteries. In anaesthetized animals, these vascular effects manifest as a selective constriction of cranial arterio-venous anastomoses resulting in a redistribution of carotid arterial blood flow. This effect is produced without significant effects on heart rate, blood pressure or blood flow to the brain, heart or lungs. Zolmitriptan also inhibits trigeminal-evoked increases in cerebral blood flow in anaesthetized cats and blocks trigeminal-evoked plasma protein extravasation in the dura of guinea-pigs. These actions are consistent with a pre-junctional inhibition of neuropeptide release from perivascular afferents of the trigeminal nerve, as confirmed by independent studies showing that zolmitriptan blocks elevations of calcitonin-gene-related peptide in jugular venous blood during electrical stimulation of the trigeminal ganglion. In all of these effects, zolmitriptan is three to four times more potent than sumatriptan, but produces the same maximum response. Zolmitriptan crosses the intact blood-brain barrier to inhibit trigeminovascular activation in the brainstem. This was shown initially by the ability of the drug to block a brainstem reflex provoking vasoactive intestinal peptide release from the VIIth cranial (facial) nerve during trigeminal stimulation. Subsequent ex vivo autoradiography confirmed that intravenously injected [3H]zolmitriptan labels a discrete population of cells in the trigeminal nucleus caudalis (TNC) and nucleus tractus solitarius. Direct evidence for a central neuromodulatory effect of zolmitriptan was provided by electrophysiological experiments which clearly demonstrated that the drug inhibits the excitability of cells in the TNC after systemic administration. This novel preclinical profile not only distinguishes zolmitriptan from sumatriptan, but raises intriguing questions about the clinical relevance of a dual action. Studies to date show that zolmitriptan indeed modulates cranial sensory processing in humans, yet central side-effects are no different from sumatriptan. This property may account for the remarkable consistency in clinical efficacy observed in clinical trials.

scholarly journals Gene transfer to the rat kidney in vivo and ex vivo using an adenovirus vector: factors influencing transgene expression

2005 ◽  
Vol 20 (7) ◽  
pp. 1385-1391 ◽  
Author(s):  
Jun Fujishiro ◽  
Shin-ichi Takeda ◽  
Yuichi Takeno ◽  
Koichi Takeuchi ◽  
Yukiyo Ogata ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Transgene Expression ◽  
Ex Vivo ◽  
Rat Kidney ◽  
Adenovirus Vector ◽  
Factors Influencing

scholarly journals Control and Augmentation of Long-Term Plasmid Transgene ExpressionIn Vivoin Murine Muscle Tissue andEx Vivoin Patient Mesenchymal Tissue

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
David Morrissey ◽  
Jan P. van Pijkeren ◽  
Simon Rajendran ◽  
Sara A. Collins ◽  
Garrett Casey ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgene Expression ◽  
Ex Vivo ◽  
Inducible Promoter ◽  
Luciferase Expression ◽  
Mesenchymal Tissue ◽  
Adenoviral Delivery ◽  
Regulated Gene Expression

Purpose. In vivogene therapy directed at tissues of mesenchymal origin could potentially augment healing. We aimed to assess the duration and magnitude of transene expressionin vivoin mice andex vivoin human tissues.Methods.Using bioluminescence imaging, plasmid and adenoviral vector-based transgene expression in murine quadricepsin vivowas examined. Temporal control was assessed using a doxycycline-inducible system. Anex vivomodel was developed and optimised using murine tissue, and applied inex vivohuman tissue.Results. In vivoplasmid-based transgene expression did not silence in murine muscle, unlike in liver. Although maximum luciferase expression was higher in muscle with adenoviral delivery compared with plasmid, expression reduced over time. The inducible promoter cassette successfully regulated gene expression with maximum levels a factor of 11 greater than baseline. Expression was re-induced to a similar level on a temporal basis. Luciferase expression was readily detectedex vivoin human muscle and tendon.Conclusions.Plasmid constructs resulted in long-termin vivogene expression in skeletal muscle, in a controllable fashion utilising an inducible promoter in combination with oral agents. Successful plasmid gene transfection in humanex vivomesenchymal tissue was demonstrated for the first time.

scholarly journals Canine Adenovirus Vectors for Lung-Directed Gene Transfer: Efficacy, Immune Response, and Duration of Transgene Expression Using Helper-Dependent Vectors

2006 ◽  
Vol 80 (3) ◽  
pp. 1487-1496 ◽  
Author(s):  
Anne Keriel ◽  
Céline René ◽  
Chad Galer ◽  
Joseph Zabner ◽  
Eric J. Kremer
Keyword(s):  
Gene Transfer ◽  
Respiratory Tract ◽  
Neutralizing Antibodies ◽  
Transgene Expression ◽  
Viral Vectors ◽  
Ex Vivo ◽  
Adenovirus Type ◽  
Transduction Efficiency

ABSTRACT A major hurdle to the successful clinical use of some viral vectors relates to the innate, adaptive, and memory immune responses that limit the efficiency and duration of transgene expression. Some of these drawbacks may be circumvented by using vectors derived from nonhuman viruses such as canine adenovirus type 2 (CAV-2). Here, we evaluated the potential of CAV-2 vectors for gene transfer to the respiratory tract. We found that CAV-2 transduction was efficient in vivo in the mouse respiratory tract, and ex vivo in well-differentiated human pulmonary epithelia. Notably, the in vivo and ex vivo efficiency was poorly inhibited by sera from mice immunized with a human adenovirus type 5 (HAd5, a ubiquitous human pathogen) vector or by human sera containing HAd5 neutralizing antibodies. Following intranasal instillation in mice, CAV-2 vectors also led to a lower level of inflammatory cytokine secretion and cellular infiltration compared to HAd5 vectors. Moreover, CAV-2 transduction efficiency was increased in vitro in human pulmonary cells and in vivo in the mouse respiratory tract by FK228, a histone deacetylase inhibitor. Finally, by using a helper-dependent CAV-2 vector, we increased the in vivo duration of transgene expression to at least 3 months in immunocompetent mice without immunosuppression. Our data suggest that CAV-2 vectors may be efficient and safe tools for long-term clinical gene transfer to the respiratory tract.

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