PlGF Repairs Myocardial Ischemia through Mechanisms of Angiogenesis, Cardioprotection and Recruitment of Myo-Angiogenic Competent Marrow Progenitors

Despite preclinical success in regenerating and revascularizing the infarcted heart using angiogenic growth factors or bone marrow (BM) cells, recent clinical trials have revealed less benefit from these therapies than expected.We explored the therapeutic potential of myocardial gene therapy of placental growth factor (PlGF), a VEGF-related angiogenic growth factor, with progenitor-mobilizing activity.Myocardial PlGF gene therapy improves cardiac performance after myocardial infarction, by inducing cardiac repair and reparative myoangiogenesis, via upregulation of paracrine anti-apoptotic and angiogenic factors. In addition, PlGF therapy stimulated Sca-1(+)/Lin(-) (SL) BM progenitor proliferation, enhanced their mobilization into peripheral blood, and promoted their recruitment into the peri-infarct borders. Moreover, PlGF enhanced endothelial progenitor colony formation of BM-derived SL cells, and induced a phenotypic switch of BM-SL cells, recruited in the infarct, to the endothelial, smooth muscle and cardiomyocyte lineage.Such pleiotropic effects of PlGF on cardiac repair and regeneration offer novel opportunities in the treatment of ischemic heart disease

Directed self-assembly of a xenogeneic vascularized endocrine pancreas for type 1 diabetes.

Intrahepatic islet transplantation is the standard cell therapy for β cell replacement. However, the shortage of organ donors and an unsatisfactory engraftment limit its application to a selected patients with type 1 diabetes. There is an urgent need to identify alternative strategies based on an unlimited source of insulin producing cells and innovative scaffolds to foster cell interaction and integration to orchestrate physiological endocrine function. We previously proposed the use of decellularized lung as a scaffold for β cell replacement with the final goal of engineering a vascularized endocrine organ. Here, we prototyped this technology with the integration of neonatal porcine islet and healthy subject-derived blood outgrowth endothelial cells to engineer a xenogeneic vascularized endocrine pancreas. We validated ex vivo cell integration and function, its engraftment and performance in a preclinical model of diabetes. Results showed that this technology not only is able to foster neonatal pig islet maturation in vitro, but also to perform in vivo immediately upon transplantation and for over 18 weeks, compared to normal performance within 8 weeks in various state of the art preclinical models. Given the recent progress in donor pig genetic engineering, this technology may enable the assembly of immune-protected functional endocrine organs

МЕТОДЫ ФОТОНИКИ ДЛЯ ОЦЕНКИ КАЧЕСТВА ПРИЖИВЛЕНИЯ КОЖНЫХ ТРАНСПЛАНТАТОВ

 In this review, based on more than 70 articles of Russian and foreign authors, methods of skin engraftment monitoring are discussed. Main processes occurring in skin on cellular and subcellular levels at different stages of engraftment are considered. Optical methods which allow performing non-invasive analysis of blood vessels, collagen concentration and form of cellular respiration (by NADH fluorescence) are described. Comparative analysis of nuclear and optical methods for engraftment monitoring highly developed and widespread nowadays is presented. The advantages of optical methods includes multifunctionality, usability and clarity of results, safety and low cost. In contrast to X-ray CT, MRI and ultrasound, optical methods can be used in monitoring mode. One of the promising directions for improving quality of engraftment due to antibacterial effect, photodynamic therapy, is described in details. The use of crystalline organic nanophotosensitizers (particularly aluminum phthalocyanine) is shown to be the most promising. The main distinctive feature of its application is that nanoparticles injected into wound surface or contact area of tissue graft are not photoactive until the moment the inflammation starts. The development of method for assessing skin condition by spectroscopic properties of tissue components (using fluorescent dyes and photosensitizers in molecular and nanoforms), which allows analyzing physiological state of skin (degree and rate of engraftment or rejection) and controlling certain biochemical and physiological parameters of a tissue graft or an entire area of affected skin is shown to be crucial.Настоящий обзор, основанный более чем на 70 проанализированных статьях российских и зарубежных авторов, посвящен методам мониторинга состояния трансплантируемых участков кожи. В обзоре рассмотрены основные процессы, происходящие в коже на клеточном и субклеточном уровнях на разных этапах приживления трансплантата. Описаны оптические методы, позволяющие проводить неинвазивный анализ состояния кровеносных сосудов, концентрации коллагена, типа клеточного дыхания (по флуоресценции NADH). Приведена сравнительная таблица ядерных и оптических методов мониторинга состояния приживления трансплантата, наиболее широко развитых и использующихся на сегодняшний день. Отмечено, что преимущество оптических методов заключается в многофункциональности, простоте использования и интерпретации результатов, безопасности, низкой стоимости, а также в возможности использования в режиме мониторинга, в отличие от рентгено-компьютерной томографии, МРТ и УЗИ. Подробно рассмотрено одно из перспективных направлений, используемых для улучшения качества приживления трансплантатов преимущественно за счет антимикробного эффекта – фотодинамическая терапия. Отмечено, что перспективным является использование для фотодинамической терапии кристаллических наночастиц органических фотосенсибилизаторов, в частности фталоцианина алюминия. Основная отличительная особенность их использования заключается в том, что наночастицы, введенные в область раневой поверхности или в контактную область приживляемой ткани или трансплантата, не проявляют фотоактивности до момента начала воспаления. Сделан вывод, что актуальной является задача по разработке метода оценки состояния кожного покрова по спектроскопическим свойствам компонентов тканей (с использованием флуоресцентных красителей и фотосенсибилизаторов в молекулярной и наноформах), который позволит анализировать физиологическое состояние кожного покрова (степень и скорость приживления или отторжения), а также контролировать некоторые биохимические и физиологические параметры трансплантата или всей области поражения кожи

Activated N-Ras signaling regulates arterial-venous specification in zebrafish

Background: The aberrant activation of Ras signaling is associated with human diseases including hematological malignancies and vascular disorders. So far the pathological roles of activated Ras signaling in hematopoiesis and vasculogenesis are largely unknown. Methods: A conditional Cre/loxP transgenic strategy was used to mediate the specific expression of a constitutively active form of human N-Ras in zebrafish endothelial and hematopoietic cells driven by the zebrafish lmo2 promoter. The expression of hematopoietic and endothelial marker genes was analyzed both via whole mount in situ hybridization (WISH) assay and real-time quantitative PCR (qPCR). The embryonic vascular morphogenesis was characterized both by living imaging and immunofluorescence on the sections with a confocal microscopy, and the number of endothelial cells in the embryos was quantified by flow cytometry. The functional analyses of the blood circulation were carried out by fluorescence microangiography assay and morpholino injection. Results: In the activated N-Ras transgenic embryos, the primitive hematopoiesis appeared normal, however, the definitive hematopoiesis of these embryos was completely absent. Further analysis of endothelial cell markers confirmed that transcription of arterial marker ephrinB2 was significantly decreased and expression of venous marker flt4 excessively increased, indicating the activated N-Ras signaling promotes the venous development at the expense of arteriogenesis during zebrafish embryogenesis. The activated N-Ras-expressing embryos showed atrophic axial arteries and expansive axial veins, leading to no definitive hematopoietic stem cell formation, the blood circulation failure and subsequently embryonic lethality. Conclusions: Our studies revealed for the first time that activated N-Ras signaling during the endothelial differentiation in vertebrates can disrupt the balance of arterial-venous specification, thus providing new insights into the pathogenesis of the congenital human vascular disease and tumorigenic angiogenesis

Exposure to the natural alkaloid Berberine affects cardiovascular system morphogenesis and functionality during zebrafish development

The plant-derived natural alkaloid berberine displays therapeutic potential to treat several pathological conditions, including dyslipidemias, diabetes and cardiovascular disorders. However, data on berberine effects during embryonic development are scarce and in part controversial. In this study, using zebrafish embryos as vertebrate experimental model, we address the effects of berberine treatment on cardiovascular system development and functionality. Starting from the observation that berberine induces developmental toxicity and pericardial edema in a time- and concentration-dependent manner, we found that treated embryos display cardiac looping defects and, at later stages, present an abnormal heart characterized by a stretched morphology and atrial endocardial/myocardial detachment. Furthermore, berberine affected cardiac functionality of the embryos, promoting bradycardia and reducing the cardiac output, the atrial shortening fraction percentage and the atrial stroke volume. We also found that, during development, berberine interferes with the angiogenic process, without altering vascular permeability. These alterations are associated with increased levels of vascular endothelial growth factor aa (vegfaa) mRNA, suggesting an important role for Vegfaa as mediator of berberine-induced cardiovascular defects. Altogether, these data indicate that berberine treatment during vertebrate development leads to an impairment of cardiovascular system morphogenesis and functionality, suggesting a note of caution in its use during pregnancy and lactation

Scanning Electron Microscopy of Corrosion Casting in Medicine

The aims of this review are: 1. to provide a bibliography of the publications that have used the corrosion casting technique; 2. to describe the advantages and limitations of the methodology; 3. to illustrate possible applications in the field of medicine, and 4. to highlight the significance of this method in the teaching of medical students. Thus, this paper is primarily focused on the scanning electron microscopical examination of vascular corrosion casts. The unsurpassed three-dimensionality of the corrosion casting technique compared to any other means stands out in particular. This can be especially useful when complex vascular-anatomical relationships are present. This applies not only to the portrayal of the modes of branching and varying vascular densities but also to regulatory arrangements, such as sphincters and arteriovenous anastomoses. Between 1966 and 1990, a total of 549 publications were found in the Medline literature data bank, containing the key words corrosion casting , microvascular cast , or vascular cast (as of August, 1990). Of those publications, most dealt with applications to experimental animals. By contrast, only 142 reports were mainly or partially concerned with human investigational material. The normal vascular system of nearly all organs, insofar as this is of direct medical relevance, has been largely resolved. In our opinion, one of the most important potential applications of the corrosion casting technique lies in the investigation of gastrointestinal, renal or hepatic ailments, which coincide with the reconstruction or rarefication of the vascular bed, e.g., in ulcers, ileitis terminalis, colitis ulcerosa, cirrhosis or glomerulonephritis

ZEBRAFISH (DANIO RERIO) AS A MODEL FOR STUDYING CARDIOVASCULAR SYSTEM DEVELOPMENT. CHARACTERIZATION OF NEW MARKERS: VE-PTP, NUMB AND NUMBLIKE.

In the last years zebrafish (Danio rerio, Teleostea) has been well established as a great model organism for comparative studies of cardiovascular embryonic development in Teleostea and Vertebrate. A lot of processes are involved in the formation of a correct vascular system and we focalized our attention on the correct assembly of endothelial junctions and on the acquisition of the arterial-venous identity of endothelial cells. Association/disassociation of endothelial cell junctions plays a decisive role in controlling vascular development and blood vessels permeability. Ve-ptp (Vascular Endothelial-Protein Tyrosine Phosphatase) is a transmembrane protein exclusively present at endothelial adherens junctions (AJs) and its potential structural role in vascular integrity is not thoroughly described in vivo. In this work we investigated the role of Ve-ptp in AJs maturation/stability and in modulation of endothelial permeability. In fact, the performed ultrastructural analysis revealed a statistically significant reduction of junction complexes and the presence of immature AJs in zve-ptp morphants but not in control embryos. Moreover alteration of the zVe-ptp extracellular domain, induced by a splicing morpholino, resulted in permeability defects, showed by microangiography assays. Arterial-venous identity determination of endothelial cells represents a critical stage during cardiovascular development. Numb is a membrane-associated protein identified in all Vertebrates, while Numblike is a cytosolic protein, homologue to Numb, first characterized in mice. The functions of Numb and Numblike are related with their ability to physically interact with Notch, working as antagonist of this protein. Notch proteins are evolutionary conserved trans-membrane receptors, which regulate several mechanisms involved in developmental processes, including neurogenesis, haematopoiesis, vasculogenesis and angiogenesis. In this work we conducted a thorough functional characterization of numb and numblike in zebrafish embryos. As a consequence of loss-of-function experiments it was detected an evident reduction in size of caudal vascular plexus (CV) and an approach of the main axial vessels (Dorsal Aorta, DA, and Posterior Cardinal Vein, PCV), defects corroborated by morphological analysis of plastic semi-thin sections. Afterwards, expression pattern analysis of specific vascular markers revealed an increase of arterial markers\u2019 expression and a decrease of venous markers\u2019 expression, proving the alteration of AV identity acquisition of vessels. All these data taken together confirm a decisive role of ve-ptp, numb and numblike during vascular development in zebrafish

Mechanistic illustration: How newly‐formed blood vessels stopped by the mineral blocks of bone substitutes can be avoided by using innovative combined therapeutics

One major limitation for the vascularization of bone substitutes used for filling is the presence of mineral blocks. The newly-formed blood vessels are stopped or have to circumvent the mineral blocks, resulting in inefficient delivery of oxygen and nutrients to the implant. This leads to necrosis within the implant and to poor engraftment of the bone substitute. The aim of the present study is to provide a bone substitute currently used in the clinic with suitably guided vascularization properties. This therapeutic hybrid bone filling, containing a mineral and a polymeric component, is fortified with pro-angiogenic smart nano-therapeutics that allow the release of angiogenic molecules. Our data showed that the improved vasculature within the implant promoted new bone formation and that the newly-formed bone swapped the mineral blocks of the bone substitutes much more efficiently than in non-functionalized bone substitutes. Therefore, we demonstrated that our therapeutic bone substitute is an advanced therapeutical medicinal product, with great potential to recuperate and guide vascularization that is stopped by mineral blocks, and can improve the regeneration of critical-sized bone defects. We have also elucidated the mechanism to understand how the newly-formed vessels can no longer encounter mineral blocks and pursue their course of vasculature, giving our advanced therapeutical bone filling great potential to be used in many applications, by combining filling and nano-regenerative medicine that currently fall short because of problems related to the lack of oxygen and nutrients