A gene transfer approach, based on Adeno-Associated Viral (AAV) vectors, to study the process of vessel maturation and stabilization

2007/2008The main goal of angiogenic gene therapy is the formation of functional new blood vessels adequate to restore blood flow in ischemic tissues. Angiogenesis is a complex process, consisting in the sprouting of new capillaries from pre-existing vessels to form an immature vascular network, which subsequently undergoes functional maturation and remodelling. Many factors are involved in this process and, among them, the VEGF family members are universally recognized as the key players. During my PhD I exploited gene transfer by vectors based on the Adeno-Associated Virus (AAV) to express several factors involved in the angiogenic process, in an attempt to define the molecular and cellular mechanisms of vessel maturation and stabilization. Most experiments were performed by vector injection in the mouse and rat skeletal muscle, followed by detailed histological, immunohistochemical and functional analysis. First of all the angiogenic effect driven by two main VEGF isoforms, VEGF165 and VEGF121 was compared. AAV-VEGF165 and AAV-VEGF121 appeared equally able to induce endothelial cell proliferation, leading to the formation of new CD31 positive capillaries. However, only the longest VEGF165 isoform was capable to recruit -SMA positive cells around growing capillaries and therefore giving rise to small arteries. The acquisition of a smooth muscle cell layer can be considered as marker of vessel maturation. This was also confirmed by a permeability assay, which showed that VEGF121-induced vessels were more permeable compared to those induced by VEGF165. Interestingly, the presence of -SMA positive vessels was paralleled by the recruitment of CD11b positive mononuclear cells from the bone marrow, cells which were not recruited by VEGF121. The presence of these infiltrating cells in close proximity to the newly formed arterioles suggested their possible role in smooth muscle cell recruitment and vessel maturation. Real-time PCR allowed observing that the infiltrating CD11b positive cells expressed a cocktail of cytokines implicated in vessel maturation, such as TGF- and PDGF-B. As a proof of concept of the paracrine activity of these cells in vessel maturation, we developed an AAV-PDGF-B vector, which, when co-injected with AAV-VEGF121, was arteriogenic even in absence of cellular infiltration. Thus, the expression of PDGF-B partially substitutes for the cells observed in the muscles injected by AAV-VEGF165 to form arterial vessels. To verify the functionality of the vessels induced by AAV-VEGF165 we delivered this vector to different animal models of tissue ischemia: a flap ischemia model and an in vivo chamber for tissue engineering based on an artero-venous loop. In both the models, VEGF165 expression induced the formation of -SMA positive vessels, which turned out to improve flap survival in the flap models, and to promote the formation of new vascularized tissue in the chamber. Despite the presence of several arteries, other vessels formed by VEGF165 were abnormally enlarged and leaky, often forming vascular lacunae. This observation indicated that VEGF gene transfer might not be sufficient for the formation of a fully functional vascular network, and that other factors might be required in order to achieve functional competence of the neovessels. We observed that the combined expression of VEGF165 with Angiopoietin-1, which is known to stabilize endothelial and mural cell interactions, resulted in a significant reduction of vessel permeability and improved blood flow, as assessed by positron emission tomography (PET) and single photon emission tomography (SPECT). These findings reveal that a fine control of the expression of angiogenic factors is needed to achieve the formation of stable and functional vessels. The presence of -SMA positive cells might be considered as a first step in vessel maturation but further stabilization factors have to take part to the process in order to tighten the cell-cell junctions. Moreover, we showed that a detailed histological and functional analysis ex vivo might not be sufficient to characterized the new vasculature, requiring imaging techniques such as PET or SPECT.XX Ciclo197

Anisotropic topographies restore endothelial monolayer integrity and promote the proliferation of senescent endothelial cells

Thrombogenicity remains a major issue in cardiovascular implants (CVIs). Complete surficial coverage of CVIs by a monolayer of endothelial cells (ECs) prior to implantation represents a promising strategy but is hampered by the overall logistical complexity and the high number of cells required. Consequently, extensive cell expansion is necessary, which may eventually lead to replicative senescence. Considering that micro-structured surfaces with anisotropic topography may promote endothelialization, we investigated the impact of gratings on the biomechanical properties and the replicative capacity of senescent ECs. After cultivation on gridded surfaces, the cells showed significant improvements in terms of adherens junction integrity, cell elongation, and orientation of the actin filaments, as well as enhanced yes-associated protein nuclear translocation and cell proliferation. Our data therefore suggest that micro-structured surfaces with anisotropic topographies may improve long-term endothelialization of CVIs. Keywords: aging; anisotropy; endothelial cells; monolayer integrity; proliferation; senescence; telomere; topograph

MiR-320a as a Potential Novel Circulating Biomarker of Arrhythmogenic CardioMyopathy

Diagnosis of Arrhythmogenic CardioMyopathy (ACM) is challenging and often late after disease onset. No circulating biomarkers are available to date. Given their involvement in several cardiovascular diseases, plasma microRNAs warranted investigation as potential non-invasive diagnostic tools in ACM. We sought to identify circulating microRNAs differentially expressed in ACM with respect to Healthy Controls (HC) and Idiopathic Ventricular Tachycardia patients (IVT), often in differential diagnosis. ACM and HC subjects were screened for plasmatic expression of 377 microRNAs and validation was performed in 36 ACM, 53 HC, 21 IVT. Variable importance in data partition was estimated through Random Forest analysis and accuracy by Receiver Operating Curves. Plasmatic miR-320a showed 0.53\u2009\ub1\u20090.04 fold expression difference in ACM vs. HC (p\u2009<\u20090.01). A similar trend was observed when comparing ACM (n\u2009=\u200913) and HC (n\u2009=\u200917) with athletic lifestyle, a ACM precipitating factor. Importantly, ACM patients miR-320a showed 0.78\u2009\ub1\u20090.05 fold expression change vs. IVT (p\u2009=\u20090.03). When compared to non-invasive ACM diagnostic parameters, miR-320a ranked highly in discriminating ACM vs. IVT and it increased their accuracy. Finally, miR-320a expression did not correlate with ACM severity. Our data suggest that miR-320a may be considered a novel potential biomarker of ACM, specifically useful in ACM vs. IVT differentiation

Inducible adeno-associated virus vectors promote functional angiogenesis in adult organisms via regulated vascular endothelial growth factor expression

AIMS: Members of the vascular endothelial growth factor (VEGF) family are among the most promising cytokines to induce neovascularization of ischaemic tissues; however, their unregulated expression often results in major undesired effects. Here, we describe the properties of inducible vectors based on the adeno-associated virus (AAV), allowing precise control of VEGF expression, and exploit these vectors to define the kinetics of the angiogenic response elicited by the factor. METHODS AND RESULTS: Based on a tetracycline-inducible transactivator, we designed an AAV vector system allowing the pharmacological regulation of VEGF production in vivo and tested its efficacy in inducing functional neoangiogenesis in both normoperfused and ischaemic skeletal muscle in mice by a combination of histological, immunofluorescent, and molecular imaging techniques. We observed that a prolonged expression of VEGF was required to determine the formation of stable vessels, able to persist upon withdrawal of the angiogenic stimulus. However, the vessels formed in the presence of continuous VEGF expression consisted mainly of dilated and leaky capillaries. As determined after pinhole scintigraphy, this abnormal vasculature accounted for a significant drop in functional tissue perfusion. In contrast, transient VEGF expression, followed by a period of VEGF withdrawal, allowed maintenance of functional perfusion under resting conditions and during exercise. This VEGF-inducible system was highly effective in improving vascularization and function in a hind-limb ischaemia model. CONCLUSION: Together, these results clearly indicate that the fine tuning of VEGF expression is required to achieve the formation of a stable vasculature able to sustain functional neovascularization

A free-form patterning method enabling endothelialization under dynamic flow

Endothelialization strategies aim at protecting the surface of cardiovascular devices upon their interaction with blood by the generation and maintenance of a mature monolayer of endothelial cells. Rational engineering of the surface micro-topography at the luminal interface provides a powerful access point to support the survival of a living endothelium under the challenging hemodynamic conditions created by the implant deployment and function. Surface structuring protocols must however be adapted to the complex, non-planar architecture of the target device precluding the use of standard lithographic approaches. Here, a novel patterning method, harnessing the condensation and evaporation of water droplets on a curing liquid elastomer, is developed to introduce arrays of microscale wells on the surface of a biocompatible silicon layer. The resulting topographies support the in vitro generation of mature human endothelia and their maintenance under dynamic changes of flow direction or magnitude, greatly outperforming identical, but flat substrates. The structuring approach is additionally demonstrated on non-planar interfaces yielding comparable topographies. The intrinsically freeform patterning is therefore compatible with a complete and stable endothelialization of complex luminal interfaces in cardiovascular implants

AAV vector encoding human VEGF–transduced pectineus muscular flaps increase the formation of new tissue through induction of angiogenesis in an in vivo chamber for tissue engineering: A technique to enhance tissue and vessels in microsurgically engineered tissue

In regenerative medicine, new approaches are required for the creation of tissue substitutes, and the interplay between different research areas, such as tissue engineering, microsurgery and gene therapy, is mandatory. In this article, we report a modification of a published model of tissue engineering, based on an arterio-venous loop enveloped in a cross-linked collagen–glycosaminoglycan template, which acts as an isolated chamber for angiogenesis and new tissue formation. In order to foster tissue formation within the chamber, which entails on the development of new vessels, we wondered whether we might combine tissue engineering with a gene therapy approach. Based on the well-described tropism of adeno-associated viral vectors for post-mitotic tissues, a muscular flap was harvested from the pectineus muscle, inserted into the chamber and transduced by either AAV vector encoding human VEGF 165 or AAV vector expressing the reporter gene β-galactosidase, as a control. Histological analysis of the specimens showed that muscle transduction by AAV vector encoding human VEGF 165 resulted in enhanced tissue formation, with a significant increase in the number of arterioles within the chamber in comparison with the previously published model. Pectineus muscular flap, transduced by adeno-associated viral vectors, acted as a source of the proangiogenic factor vascular endothelial growth factor, thus inducing a consistent enhancement of vessel growth into the newly formed tissue within the chamber. In conclusion, our present findings combine three different research fields such as microsurgery, tissue engineering and gene therapy, suggesting and showing the feasibility of a mixed approach for regenerative medicine

Mechanical Fingerprint of Senescence in Endothelial Cells

Endothelial senescence entails alterations of the healthy cell phenotype, which accumulate over time and contribute to cardiovascular disease. Mechanical aspects regulating cell adhesion, force generation, and the response to flow contribute to the senescence-associated drift; however, they remain largely unexplored. Here, we exploit force microscopy to resolve variations of the cell anchoring to the substrate and the tractions generated upon aging in the nanonewton (nN) range. Senescent endothelial cells display a multifold increase in the levels of basal adhesion and force generation supported by mature and strong focal adhesions. The enhanced mechanical interaction with the substrate yields static endothelial monolayers that polarize in response to flow but fail the process of coordinated cell shape remodeling and reorientation. The emerging picture indicates that senescence reinforces the local cell interaction with the substrate and may therefore prevent endothelial denudation; however, it compromises the ability to functionally adapt to the local hemodynamic conditions.ISSN:1530-6984ISSN:1530-699

miR-200 family members reduce senescence and restore idiopathic pulmonary fibrosis type II alveolar epithelial cell transdifferentiation

Rationale: Alveolar type II (ATII) cells act as adult stem cells contributing to alveolar type I (ATI) cell renewal and play a major role in idiopathic pulmonary fibrosis (IPF), as supported by familial cases harbouring mutations in genes specifically expressed by these cells. During IPF, ATII cells lose their regenerative potential and aberrantly express pathways contributing to epithelial\u2013mesenchymal transition (EMT). The microRNA miR-200 family is downregulated in IPF, but its effect on human IPF ATII cells remains unproven. We wanted to 1) evaluate the characteristics and transdifferentiating ability of IPF ATII cells, and 2) test whether miR-200 family members can rescue the regenerative potential of fibrotic ATII cells. Methods: ATII cells were isolated from control or IPF lungs and cultured in conditions promoting their transdifferentiation into ATI cells. Cells were either phenotypically monitored over time or transfected with miR-200 family members to evaluate the microRNA effect on the expression of transdifferentiation, senescence and EMT markers. Results: IPF ATII cells show a senescent phenotype ( p16 and p21), overexpression of EMT (ZEB1/2) and impaired expression of ATI cell markers (AQP5 and HOPX) after 6 days of culture in differentiating medium. Transfection with certain miR-200 family members (particularly miR-200b-3p and miR-200c-3p) reduced senescence marker expression and restored the ability to transdifferentiate into ATI cells. Conclusions: We demonstrated that ATII cells from IPF patients express senescence and EMT markers, and display a reduced ability to transdifferentiate into ATI cells. Transfection with certain miR-200 family members rescues this phenotype, reducing senescence and restoring transdifferentiation marker expression

Improved Survival of Ischemic Cutaneous and Musculocutaneous Flaps after Vascular Endothelial Growth Factor Gene Transfer Using Adeno-Associated Virus Vectors

A major challenge in reconstructive surgery is flap ischemia, which might benefit from induction of therapeutic angiogenesis. Here we demonstrate the effect of an adeno-associated virus (AAV) vector delivering vascular endothelial growth factor (VEGF)165 in two widely recognized in vivo flap models. For the epigastric flap model, animals were injected subcutaneously with 1.5 × 10(11) particles of AAV-VEGF at day 0, 7, or 14 before flap dissection. In the transverse rectus abdominis musculocutaneous flap model, AAV-VEGF was injected intramuscularly. The delivery of AAV-VEGF significantly improved flap survival in both models, reducing necrosis in all treatment groups compared to controls. The most notable results were obtained by administering the vector 14 days before flap dissection. In the transverse rectus abdominis musculocutaneous flap model, AAV-VEGF reduced the necrotic area by >50% at 1 week after surgery, with a highly significant improvement in the healing process throughout the following 2 weeks. The therapeutic effect of AAV-VEGF on flap survival was confirmed by histological evidence of neoangiogenesis in the formation of large numbers of CD31-positive capillaries and α-smooth muscle actin-positive arteriolae, particularly evident at the border between viable and necrotic tissue. These results underscore the efficacy of VEGF-induced neovascularization for the prevention of tissue ischemia and the improvement of flap survival in reconstructive surgery

Improved survival of ischemic cutaneous and musculocutaneous flaps after vascular endothelial growth factor gene transfer using adeno-associated virus vectors

A major challenge in reconstructive surgery is flap ischemia, which might benefit from induction of therapeutic angiogenesis. Here we demonstrate the effect of an adeno-associated virus (AAV) vector delivering vascular endothelial growth factor (VEGF)165 in two widely recognized in vivo flap models. For the epigastric flap model, animals were injected subcutaneously with 1.5 X 10(11) particles of AAV-VEGF at day 0, 7, or 14 before flap dissection. In the transverse rectus abdominis musculocutaneous flap model, AAV-VEGF was injected intramuscularly. The delivery of AAV-VEGF significantly improved flap survival in both models, reducing necrosis in all treatment groups compared to controls. The most notable results were obtained by administering the vector 14 days before flap dissection. In the transverse rectus abdominis musculocutaneous flap model, AAV-VEGF reduced the necrotic area by &gt; 50% at 1 week after surgery, with a highly significant improvement in the healing process throughout the following 2 weeks. The therapeutic effect of AAV-VEGF on flap survival was confirmed by histological evidence of neoangiogenesis in the formation of large numbers of CD31-positive capillaries and alpha-smooth muscle actin-positive arteriolae, particularly evident at the border between viable and necrotic tissue. These results underscore the efficacy of VEGF-induced neovascularization for the prevention of tissue ischemia and the improvement of flap survival in reconstructive surgery