The use of vectors based on the small parvovirus adeno-associated virus has gained significant momentum during the past decade. Their high efficiency of transduction of postmitotic tissues in vivo, ...such as heart, brain, and retina, renders these vectors extremely attractive for several gene therapy applications affecting these organs. Besides functional correction of different monogenic diseases, the possibility to drive efficient and persistent transgene expression in the heart offers the possibility to develop innovative therapies for prevalent conditions, such as ischemic cardiomyopathy and heart failure. Therapeutic genes are not only restricted to protein-coding complementary DNAs but also include short hairpin RNAs and microRNA genes, thus broadening the spectrum of possible applications. In addition, several spontaneous or engineered variants in the virus capsid have recently improved vector efficiency and expanded their tropism. Apart from their therapeutic potential, adeno-associated virus vectors also represent outstanding investigational tools to explore the function of individual genes or gene combinations in vivo, thus providing information that is conceptually similar to that obtained from genetically modified animals. Finally, their single-stranded DNA genome can drive homology-directed gene repair at high efficiency. Here, we review the main molecular characteristics of adeno-associated virus vectors, with a particular view to their applications in the cardiovascular field.
Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder in which learning, memory and cognitive functions decline progressively. Familial forms of AD (FAD) are caused by ...mutations in amyloid precursor protein (
), presenilin 1 (
) and presenilin 2 (
) genes. Presenilin 1 (PS1) and its homologue, presenilin 2 (PS2), represent, alternatively, the catalytic core of the γ-secretase complex that, by cleaving APP, produces neurotoxic amyloid beta (Aβ) peptides responsible for one of the histopathological hallmarks in AD brains, the amyloid plaques. Recently,
FAD mutations have been associated with a loss-of-function phenotype. To investigate whether this finding can also be extended to
FAD mutations, we studied two processes known to be modulated by PS2 and altered by FAD mutations: Ca
signaling and mitochondrial function. By exploiting neurons derived from a
knock-out (PS2-/-) mouse model, we found that, upon IP
-generating stimulation, cytosolic Ca
handling is not altered, compared to wild-type cells, while mitochondrial Ca
uptake is strongly compromised. Accordingly, PS2-/- neurons show a marked reduction in endoplasmic reticulum-mitochondria apposition and a slight alteration in mitochondrial respiration, whereas mitochondrial membrane potential, and organelle morphology and number appear unchanged. Thus, although some alterations in mitochondrial function appear to be shared between PS2-/- and FAD-PS2-expressing neurons, the mechanisms leading to these defects are quite distinct between the two models. Taken together, our data appear to be difficult to reconcile with the proposal that FAD-PS2 mutants are loss-of-function, whereas the concept that PS2 plays a key role in sustaining mitochondrial function is here confirmed.
We have previously shown that VEGF165 gene delivery into ischemic skeletal muscle exerts not only proangiogenic, but also remarkable antiapoptotic and proregenerative activity. The aim of this study ...was to determine whether recombinant adeno-associated virus (rAAV)-mediated gene delivery of VEGF165 into cardiac muscle, during acute myocardial infarction, exerts a protective effect to promote long-term functional recovery. Acute infarction of the anterior LV wall was induced in 12 chronically instrumented dogs by permanent occlusion of the LAD coronary artery. Four hours after occlusion, rAAV-VEGF165 or rAAV-LacZ (n=6 each; 5x10(12) viral particles per animal) was directly injected with an echo-guided needle into the dysfunctional cardiac wall. LV and arterial pressure, dP/dtmax, and ejection fraction were not significantly different between the two groups over time. In contrast, in the infarcted region, at four weeks after infarction, fractional shortening was 75+/-18% and -3+/-15% of baseline and length-pressure area was 54+/-15% and 0.8+/-15% of baseline in VEGF165 versus LacZ, respectively (P<0.05). Histological analysis of the border regions showed a marked increase in the number of alpha-SMA-positive arterioles (68+/-2.8 versus 100+/-3.8 vessels per microscopic field in LacZ and VEGF165 group, respectively; P<0.05). In both groups, the receptor VEGFR-2 was diffusely expressed on the surviving cardiomyocytes and, consistently, myocardial viability was significantly improved in the VEGF165-treated group, with several troponin T-expressing cardiomyocytes displaying nuclear positivity for the proliferation marker PCNA. Altogether, our results indicate that VEGF165 gene delivery exerts a marked beneficial action by enhancing both arteriologenesis and cardiomyocyte viability in infarcted myocardium.
The budding yeast Saccharomyces cerevisiae supports replication of many different RNA or DNA viruses (e.g. Tombusviruses or Papillomaviruses) and has provided means for up-scalable, cost- and ...time-effective production of various virus-like particles (e.g. Human Parvovirus B19 or Rotavirus). We have recently demonstrated that S. cerevisiae can form single stranded DNA AAV2 genomes starting from a circular plasmid. In this work, we have investigated the possibility to assemble AAV capsids in yeast.
To do this, at least two out of three AAV structural proteins, VP1 and VP3, have to be simultaneously expressed in yeast cells and their intracellular stoichiometry has to resemble the one found in the particles derived from mammalian or insect cells. This was achieved by stable co-transformation of yeast cells with two plasmids, one expressing VP3 from its natural p40 promoter and the other one primarily expressing VP1 from a modified AAV2 Cap gene under the control of the inducible yeast promoter Gal1. Among various induction strategies we tested, the best one to yield the appropriate VP1:VP3 ratio was 4.5 hour induction in the medium containing 0.5% glucose and 5% galactose. Following such induction, AAV virus like particles (VLPs) were isolated from yeast by two step ultracentrifugation procedure. The transmission electron microscopy analysis revealed that their morphology is similar to the empty capsids produced in human cells.
Taken together, the results show for the first time that yeast can be used to assemble AAV capsid and, therefore, as a genetic system to identify novel cellular factors involved in AAV biology.
The mechanisms underlying the increased cardiovascular risk after menopause are poorly understood. Estrogens modulate the cardiac renin-angiotensin system (RAS) and influence cardiac adaptation to ...afterload. To investigate whether the loss of the natural inhibition of the RAS by estrogen may be linked to an increase of cardiac apoptosis, we studied 17β-estradiol (E2) and/or angiotensin-converting enzyme (ACE) inhibitor treatment effects on cardiomyocyte survival in ovariectomized spontaneously hypertensive rats (SHRs).
Five groups of female SHRs were evaluated for 8 weeks. One group served as nonovariectomized control; the other four groups underwent bilateral ovariectomy and were randomized to receive 60-day-release pellets containing placebo or 0.5 mg of E2, the ACE inhibitor ramipril at the dosage of 2.5 mg/kg per day, or the combination of the two treatments.
Ovariectomy increased cardiomyocyte apoptosis and induced proapoptotic changes of Bcl-2 and Bax genes and proteins. These modifications were associated with an upregulation of ACE and angiotensin II type 1 (AT1) receptor genes. Ramipril was as effective as E2 in preventing cardiac apoptosis and in restoring cardiac brain natriuretic peptide in association with reduced cardiac ACE and AT1 receptor gene expression. In contrast to the ramipril treatment, the favorable effect of E2 on cardiac apoptosis occurred independently from changes in SBP. No synergistic effect was observed when the two treatments were combined.
These data show that ovariectomy stimulates myocardium apoptosis by a mechanism involving Bax and Bcl-2 genes. The antiapoptotic effect of E2 and ACE inhibitor treatment was linked to a downregulation of cardiac RAS.
Genetically Encoded Ca
2+
Indicators (GECIs) are extensively used to study organelle Ca
2+
homeostasis, although some available probes are still plagued by a number of problems, e.g., low ...fluorescence intensity, partial mistargeting, and pH sensitivity. Furthermore, in the most commonly used mitochondrial Förster Resonance Energy Transfer based-GECIs, the donor protein ECFP is characterized by a double exponential lifetime that complicates the fluorescence lifetime analysis. We have modified the cytosolic and mitochondria-targeted Cameleon GECIs by (1) substituting the donor ECFP with mCerulean3, a brighter and more stable fluorescent protein with a single exponential lifetime; (2) extensively modifying the constructs to improve targeting efficiency and fluorescence changes caused by Ca
2+
binding; and (3) inserting the cDNAs into adeno-associated viral vectors for
in vivo
expression. The probes have been thoroughly characterized
in situ
by fluorescence microscopy and Fluorescence Lifetime Imaging Microscopy, and examples of their
ex vivo
and
in vivo
applications are described.
•
Donor substitution in a mitochondrial Ca
2+
sensor improves photo-physical properties
•
Mitochondria-targeting sequence amelioration enhances the sensor localization
•
Donor substitution allows FLIM-FRET analysis, with a compensation for pH bias
•
The performance of the sensor is improved
in situ
,
ex vivo
, and
in vivo
Biological Sciences Tools; Cell Biology; Optical Imaging
Ang-1 (angiopoietin-1) improves the ineffective angiogenesis and impaired wound healing in diabetes; however, the mechanism underlying this positive effect is still far from being completely ...understood. In the present study, we investigated whether rAAV (recombinant adeno-associated virus)-Ang-1 gene transfer could improve wound repair in genetically diabetic mice (db+/db+) and the mechanism(s) by which it causes new vessel formation. An incisional skin-wound model in diabetic and normoglycaemic mice was used. After the incision, animals received rAAV-LacZ or rAAV-Ang-1 in the wound edge. After 7 and 14 days, wounds were used to (i) confirm Ang-1 gene transfer, (ii) assess histologically the healing process, (iii) evaluate wound-breaking strength, and (iv) study new vessel formation by PECAM-1 (platelet/endothelial cell adhesion molecule-1) immunostaining. Finally, we investigated VEGF (vascular endothelial growth factor) mRNA and protein levels, eNOS (endothelial NO synthase) expression and VEGFR-1 and VEGFR-2 (VEGF receptor-1 and -2 respectively) immunostaining. The efficiency of Ang-1 gene transfer was confirmed by increased mRNA and protein expression of the protein. rAAV-Ang-1 significantly improved the healing process, stimulating re-epithelization and collagen maturation, increasing breaking strength and significantly augmenting the number of new vessels, as indicated by PECAM-1 immunostaining. However, Ang-1 gene transfer did not modify the decrease in VEGF mRNA and protein expression in diabetic mice; in contrast, Ang-1 increased eNOS expression and augmented nitrate wound content and VEGFR-2 immunostaining and protein expression. Ang-1 gene transfer did not change vascular permeability. Similar results were obtained in normoglycaemic animals. In conclusion, our results provide strong evidence that Ang-1 gene transfer improves the delayed wound repair in diabetes by inducing angiogenesis in a VEGF-independent manner.
Vectors based on the adeno-associated virus (AAV) deliver therapeutic genes to muscle and heart at high efficiency and maintain transgene expression for long periods of time. Here we report about the ...synergistic effect on blood vessel formation of AAV vectors expressing the 165 aa isoform of vascular endothelial growth factor (VEGF165), a powerful activator of endothelial cells, and of angiopoietin-1 (Ang-1), which is required for vessel maturation. High titer AAV-VEGF165 and AAV-Ang-1 vector preparations were injected either alone or in combination in the normoperfused tibialis anterior muscle of rats. Long term expression of VEGF165 determined massive cellular infiltration of the muscle tissues over time, with the formation of a large set of new vessels. Strikingly, some of the cells infiltrating the treated muscles were found positive for markers of activated endothelial precursors (VEGFR-2/KDR and Tie-2) and for c-kit, an antigen expressed by pluripotent bone marrow stem cells. Expression of VEGF165 eventually resulted in the formation of structured vessels surrounded by a layer of smooth muscle cells. Presence of these arteriolae correlated with significantly increased blood perfusion in the injected areas. Co-expression of VEGF165 with angiopoietin-1-which did not display angiogenic effect per se-remarkably reduced leakage of vessels produced by VEGF165 alone.
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