In general, hitherto, all scholarly illustrations of oxidative phosphorylation are schematically presented in two dimensions as in Fig.1, where the electron current is shown resulting in the transmembrane proton gradient driving the formation of ATP via the F1F0- ATPase [1]. In this hypothesis, the electrical gradient that results from electron transport induces a conformational change of the F1F0-ATPase coupled with a change of hydrogen bonding of a strategic shell of H2O molecules, favouring the thermodynamics of the reaction ADP with inorganic phosphate (Pi) to produce ATP. This hypothesis presents the whole coupling process of the mitochondrial inner membrane in three dimensions, essential for this particulate biochemistry [1,2].
Bone represents a well vascularized structure which is remodelled and renewed continuously. It consists of osteoblasts, osteoclasts and osteocytes which have a precise role in the context of endochondral and intramembranous ossification. While the link between the bone tissue, bone marrow and the haematopoiesis is crucial for the generation of progenitor bone forming cells, recent research indicates that bone physiology as well as bone healing, repair and regeneration is directly dependent on bone angiogenesis. Experimental research suggest that angiogenesis and osteogenesis are directly coupled through a mechanism in which type H endothelial cells have a role of paramount importance. Apart from type H endothelial cells, other molecular elements such as HIF and Notch as well as CD31-endomucin capillaries and miR-497∼195 endothelial clusters may have a significant role in angiogenesis-osteogenesis coupling. The number of type H endothelial cells decreases significantly in elderly and is paralleled by a significant drop in the supply of progenitor cells. This would explain the bone loss and the decreased bone regeneration potential seen in elderly patients. Overall, it is suggested that endothelial cells and bone angiogenesis would represent therapeutic targets in various pathological conditions characterized by bone loss and impaired regeneration.
The family of Notch proteins plays a key role in cell fate determination. Additionally, Notch proteins regulate critical functions of the endothelium, as well as other recruited supporting cells, in concert with other pathways. Despite significant advances in the field and extensive studies focused on elucidating this pathway, many questions remain regarding Notch activation and its upstream/downstream effects, with vascular biology constituting one area of particular interest. Here, we provide a brief description of the components and functions of the Notch pathway in vasculature, followed by a detailed compilation of recommended methods of evaluation in vitro and in vivo. We provide a rationale for key elements when choosing different approaches and controls, strengths and limitations, and essential considerations when providing a meaningful interpretation of results. Our aim is to describe a careful approach to assessing Notch function in endothelial cells, based on underlying principles, with the overall goal of obtaining physiologically relevant information that will enhance our understanding of this pathway and its role in vascular biology.
Background: Cryopreserved bone allografts are often used to reconstruct segmental bone defects. They are non-viable, which can result in infection, non-union and stress-fractures. We aimed to revascularize allografts in porcine and rat models using vascular endothelial growth factor (VEGF), combined with platelet derived growth factor (PDGF) administered through an adeno-associated viral vector. We report the development of vascular tumors resulting from this treatment. Methods: In two separate studies, an identical AAV.VEGF.PDGF vector was used to promote angiogenesis in cryopreserved bone allografts. In 8 Yucatan minipigs, a 3.5 cm segmental tibial defect was reconstructed with a matched allograft, revascularized by placement of a transfected arteriovenous (AV) bundle within the medullary canal. In another experiment, cryopreserved femoral bone allografts coated with AAV.VEGF.PDGF were placed across a 10 mm segmental femoral gap in 10 Lewis rats. Results: Vascular tumors developed in skin and subcutaneous tissues in 5 out of 8 pigs and all of the rats. Histology revealed changes essentially identical to those seen in pyogenic granuloma (lobular capillary hemangioma) in humans. Polymerase chain reaction (PCR) identified the sequence of human VEGF-DNA in all of the sampled tumor tissues. Conclusion: Recombinant AAV gene therapy used to promote angiogenesis in avascular bone risks the development of vascular cutaneous lesions. Gene therapy using an identical AAV.VEGF.PDGF vector should not be considered clinically until safe use can be demonstrated and.concerns regarding chromosomal integration, dose effect and species differences are addressed.
Background: The application of Photobiomodulation (PBM), Piezoelectric Surgery (PES) and Concentrated Growth Factors (CGF) in dental implant sinus lift procedure has gained popularity. \r\nAim: To detail a two cases report on the use of PMB, PES, and CGF as a means of enhanced angiogenesis in tissue regeneration in dental implant lateral wall sinus lift procedure. \r\nMaterials and Methods: This paper presents two cases report on the multi-disciplined approach using PBM, PES, and CGF as a method of enriched angiogenesis in tissue regeneration in dental implant sinus lift technique. In both patients, the sinus lateral windows were created using PES and the sinus membrane was raised to make a new partition. Surface modified dental implants (average 8 mm long) were inserted concurrently, packed with autologous fibrin-rich CGF, and then lastly induced PBM using Multiwave Locked System (MLS) laser. Radiographic and clinical evaluation was implemented to validate the efficacy of this multi-disciplined approach. \r\nResults and discussion: Postoperative revival was ordinary with no noteworthy postoperative complications. Wound closure/angiogenesis occurred shortly after day one. New bone formation in all augmented maxillary sinuses was detected alongside the implants on plain radiographs and on cone-beam computed tomograms. The patients were very happy with the overall treatment (\u003e80%) and the success rate of implant was 100% after a usual 10 months loading period. PBM, PES, and CGF enhance angiogenesis/wound healing by speeding up wound approximation/healing, lessening the followings: postoperative pain, swelling, bleeding, speech impairment, analgesic use, and trismus. \r\nConclusion: Using PBM, PES, and CGF as a method to enrich angiogenesis/wound healing in sinus lift rendered initial phase of satisfaction, simplicity, effectiveness, minimal complication, enhanced angiogenesis/wound healing with no cross infection and allergic reaction.
Nordic Immunohistochemical Quality Control (NordiQC) performs proficiency testing for about 600 pathology laboratories in more than 50 countries. All general results are published on www.nordiqc.org. Over-all, about 30% of the staining results on circulated slides from tissue micro arrays have by an expert group been assessed as insufficient for diagnostic use. This paper describes the results from the two latest NordiQC runs for CD31. Out of 476 stains submitted, 30.5% were considered insufficient, mostly due to too weak or false negative staining reactions. The best results were obtained by use of mouse monoclonal antibody JC70A with an optimized protocol based on efficient heat induced epitope retrieval and a three-step polymer/multimer conjugate as visualization system. The mouse monoclonal antibody 1A10 gave unsatisfactory results in almost all cases.
Comorbidities like diabetes, arterial hypertension, or comorbidity factors such as hypercholesterolemia is common in elderly persons and are associated with higher risk of stroke. Since stroke afflicts mostly the elderly comorbid patients, it is highly desirable to test the efficacy of cell therapies in an appropriate animal stroke model. Animal models of stroke often ignore age and comorbidities frequently associated with aging, and this could be one of the explanations for unsuccessful bench-to-bedside translation of neuroprotective strategies. In order to mimic more closely the clinical condition, we have established a model of ischemic stroke in aged rats. Using this model, we showed that post-stroke angiogenesis is not impaired in the lesioned, aged brain and conclude that stroke in aged rodent models in reliable and more closely related to the human condition.
The Covid-19 pandemic represents an unsolved problem which has caused numerous fatalities. At the present time, there is no vaccination available or curative therapy. However, recent reports suggest that SARS-CoV2 acts upon its functional receptor ACE2 inducing a variety of deleterious effects such as inflammation, endothelial dysfunction, microangiopathy, myocarditis, thrombosis and myocardial infarction. The details of the SARS-CoV2 -ACE2 interaction are poorly understood and most of the hypothesis related to this are based on the extrapolation of previous research focusing on ACE2 as a receptor for SARS-CoV. Considering the similarities between SARS-CoV2 and SARS-CoV we have conducted e physiopathological analysis focusing on the key pathogenic role of ACE2 as a functional receptor for SARS-CoV2. In this context, we have identified several potential therapeutic targets which should be further evaluated in patients with Covid-19. It is likely that an efficient therapy for Covid-19 will be revealed by research investigating the binding of viral spike S protein to ACE2, and the immunological response determined by SARS-CoV2-ACE2 interaction, including the anti-viral role of plasmacytoid dendritic cells and anti-inflammatory reprogramming of macrophages.
Non-union of scaphoid bone fractures, though an uncommon occurance, are a signficant cause of morbidity in young patients. This review outlines the current understanding of vascularised bone grafts and non-union, and examines the most current clinical literature regarding the role of these procedures in modern orthopaedic practice. Composite evidence from level III studies would support using osseous grafts on a vascular pedicle for patients with non-union of the proximal end of the scaphoid with associated avascular necrosis and in cases of revision surgery where a previous non-vascularised graft has failed to achieve bony union.
Chicken chorioallantoic membrane (CAM) models have been applied as an affordable and efficient angiogenesis model for many years in relevant areas such as cancer angiogenesis and cardiovascular sciences. However, this model has intrinsic weaknesses such as the difficulty of operation and the limited size of the viewing windows. Efforts of trying to improve this model, such as transferring the embryos into petri dishes, has led to compromised survival rate of the embryos. In this study we improve the workflow by using surrogate egg shells. The images obtained from the viewing windows of the surrogate shells allow us to apply automated image analysis. This CAM model in surrogate shells combined with automated image analysis significantly decreases the labour whilst also increasing the data quality, when compared to traditional CAM models. Furthermore, to demonstrate the potential of our model, we have investigated the effects of a range of chylomicrons, extracted from murine and human samples, using the improved CAM model, to seek clues to elucidate the controversial roles of high level chylomicrons (CMs) in hypertriglyceridaemia (HTG) on atherosclerosis. We show that both native and modified chylomicrons (CMs) promote in vitro and in vivo angiogenesis. As angiogenesis is a risk factor in atherosclerosis our results provide a new mechanism for the effect of hypertriglyceridaemia (HTG) on atherosclerosis development. In summary, we demonstrate an improved CAM model aided by automated image analysis, we demonstrate the potential of this model by demonstrating interfered angiogenesis caused by HTG and discuss the impact this could potentially cause other pathological processes, such as atherosclerosis or carcinogenesis. Furthermore, the significantly low cost of CAM models compared with traditional murine models for cardiovascular research will have potential 3Rs (replacement, reduction and refinement) significance for the research communities, especially for laboratories with limited budgets or regulatory restrictions.
Angiogenesis or formation of new blood vessels as a consequence of "sprouting" in a pre-existent vascular network represents a phenomenon of paramount importance in physiology and pathology. Although the molecular pathways and factors which modulate angiogenesis are similar regardless the clinical context, there are numerous aspects related to this process which are incompletely understood. At the present time, significant research focuses in finding the best method to control the pro-angiogenic factors as well as anti-angiogenesis since therapy of various medical conditions requires either stimulation or inhibition of formation of new blood vessels. However, angiogenesis is different in various vascular systems and organs. In this review, we will present the most promising factors that interfere with angiogenesis suggesting new or improved therapeutic methods in various pathological settings.
One important initiative that commenced at the RCPAQAP in 2017 was collaboration with UK NEQAS ICC and ISH for the challenging implementation of a PD-L1 immunohistochemistry (IHC) proficiency testing program for non-small cell lung carcinoma. Challenges of assessment included standardising the scoring criteria for each commercial assay interpretation in addition to laboratory developed tests (LDTs) and using appropriate tissue to cover the critical interpretation points for each assay. The findings of the pre-Pilot suggest that the use of a clinically validated PD-L1 IHC assay performs better during assessment than adopting an LDT. It was acknowledged that participants are limited by the platforms they have available and so it was suggested that validating an optimal method against the clinical assay and continual verification of the test may produce the expected result. The next big challenge is to extend proficiency testing from technical to interpretation.
Zebrafish with fluorescently labelled blood vessels provide an excellent model for studying angiogenesis. Most commonly the growth of the intersegmental blood vessels is investigated in response to compounds or manipulation of gene expression and analysed using manual methods, typically scoring the connectivity of these blood vessels to the dorsal longitudinal anastomotic vessel. Such methods are laborious and best suited to time points after the connectivity of these vessels have been established. By contrast, reported image processing-based methods are difficult to implement and often depend on specialist software. This study aimed to develop and evaluate a computational method using the freely available ImageJ software to quantify the development of intersegmental blood vessel formation. This methodology developed allowed rapid analysis of vascular development. The outputs of total vessel length and number of junctions best documented defective vascular development at differing levels of severity and gave comparable results to the frequently used manual approach of calculating percentage connectivity of intersegmental vessels. This ImageJ-based method allowed objective quantitation of vascular network formation in zebrafish enabling a free, straightforward and rapid approach to determine the effect of novel compounds or genetic manipulation of the angiogenic process.
Angiogenesis is a fundamental process during development and disease, and many details of the underlying molecular and cellular mechanisms are incompletely understood. Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2), a major downstream target of p38 MAPK, has recently been identified as a regulator of Interleukin 1β dependent angiogenesis in vivo, and in vitro data suggest a role of MK2 for VEGF-dependent angiogenic processes in endothelial cells. We thus hypothesized that MK2 plays a role during physiological vascular development in vivo. Vascular development was investigated in the retina of MK2-deficient mice. Retinal angiogenesis such as sprouting, branching and pruning was unchanged in MK2-/- mice compared to wildtype littermates. Early arterial development was also comparable between genotypes. However, with further expansion of vascular smooth muscle cells (SMC) during maturation of the arterial network at later time points, the number of arterial branch points was significantly lower in MK2-/- mice, resulting in a reduced total arterial area in adult mice. Isolated aortic smooth muscle cells from MK2-/- mice showed a more dedifferentiated phenotype in vitro and downregulation of central SMC marker genes, consistent with the known impaired migration of MK2-/- SMC. In conclusion, MK2 is not required for physiological retinal angiogenesis. However, its loss is associated with an altered genetic profile of SMC and an impaired arterial network in adult mice, indicating a distinct and probably cell-specific role of MK2 in arteries.
We present the first documented case of an ectopic intramedullary hematopoietic niche within a cervical zygapophyseal joint intra-articular meniscoid. The meniscoid was identified in the cervical spine of an 81-year-old male cadaver, located at the dorsal aspect of the right C4-5 zygapophyseal joint, and was noted to be larger and more dense than other meniscoids during disarticulation and dissection of the joint. Histologically, the meniscoid contained hematopoietic cells surrounded by lamellar bone, and was lined with articular cartilage on the surface protruding into the joint cavity. Radiologically, the structure was hypointense on both T1- and T2-weighted magnetic resonance imaging sequences. The potential mechanisms underpinning development of this novel finding and the possible clinical implications are discussed in this report.
BACKGROUND:Endothelial cells (EC) grown on collagen particles inhibit intimal hyperplasia in animal models when applied perivascularly, and this effect appears to be, at least in part, the result of EC-derived soluble factors that suppress local vascular inflammation. To elucidate the molecular basis of the therapeutic effects of EC grown on collagen particles, the anti-inflammatory activity of conditioned medium from these cells was characterized.METHODS:Human aortic EC (HAEC) and, for chromatin immunoprecipitation assays, human umbilical vein EC (HUVEC) were treated with tumor necrosis factor alpha (TNFα) in the presence of conditioned medium generated by HAEC grown on collagen particles (ECPCM), and the anti-inflammatory effects were evaluated by analysing the expression of the inflammation-related adhesion molecules E-selectin and vascular cell adhesion molecule-1 (VCAM-1). The therapeutic activity of ECPCM was studied using the mouse strain JR5558, which develops spontaneous choroidal neovascularisation (CNV) lesions driven by local inflammation.RESULTS:ECPCM significantly suppressed TNFα-induced expression of E-selectin and VCAM-1. ECPCM did not affect the mRNA stability of the two genes, but suppressed TNFα-induced binding of the p65 subunit of NF-kB transcription factor to E-selectin and VCAM-1 promoters. In vivo, systemic ECPCM treatment significantly reduced the CNV area and the recruitment of activated macrophages to the lesions. Characterization of the molecule responsible for the anti-inflammatory activity in ECPCM indicates that it is unlikely to be a protein and that it is not any of the better characterized EC-derived anti-inflammatory molecules.CONCLUSIONS:Medium conditioned by HAEC grown on collagen particles exhibits significant anti-inflammatory activity via inhibition of genes that mediate inflammatory responses in EC.
BACKGROUND:Adipose-derived mesenchymal stromal cells (Ad-MSCs) are a promising tool for advanced cell-based therapies. They are routinely obtained enzymatically from fat lipoaspirate (LP) as SVF, and may undergo prolonged ex vivo expansion, with significant senescence and decline in multipotency. Besides, these techniques have complex regulatory issues, thus incurring in the compelling requirements of GMP guidelines. Hence, availability of a minimally manipulated, autologous adipose tissue would have remarkable biomedical and clinical relevance. For this reason, a new device, named Lipogems® (LG), has been developed. This ready-to-use adipose tissue cell derivate has been shown to have in vivo efficacy upon transplantation for ischemic and inflammatory diseases. To broaden our knowledge, we here investigated the angiogenic and anti-inflammatory properties of LG and its derived MSC (LG-MSCs) population.METHODS:Human LG samples and their LG-MSCs were analyzed by immunohistochemistry for pericyte, endothelial and mesenchymal stromal cell marker expression. Angiogenesis was investigated testing the conditioned media (CM) of LG (LG-CM) and LG-MSCs (LG-MSCs-CM) on cultured endothelial cells (HUVECs), evaluating proliferation, cord formation, and the expression of the adhesion molecules (AM) VCAM-1 and ICAM-1. The macrophage cell line U937 was used to evaluate the anti-inflammatory properties, such as migration, adhesion on HUVECs, and release of RANTES and MCP-1.RESULTS:Our results indicate that LG contained a very high number of mesenchymal cells expressing NG2 and CD146 (both pericyte markers) together with an abundant microvascular endothelial cell (mEC) population. Substantially, both LG-CM and LG-MSC-CM increased cord formation, inhibited endothelial ICAM-1 and VCAM-1 expression following TNFα stimulation, and slightly improved HUVEC proliferation. The addition of LG-CM and LG-MSC-CM strongly inhibited U937 migration upon stimulation with the chemokine MCP-1, reduced their adhesion on HUVECs and significantly suppressed the release of RANTES and MCP-1.CONCLUSIONS:Our data indicate that LG micro-fragmented adipose tissue retains either per se, or in its embedded MSCs content, the capacity to induce vascular stabilization and to inhibit several macrophage functions involved in inflammation.
BACKGROUND:Biological evidence reported in the literature supports the role of CELSR1 as being essential for valvular function in murine lymphatics. Yet thus far, there have been no variants in CELSR1 associated with lymphatic dysfunction in humans.CASE PRESENTATION:In this report, a rare early inactivating mutation in CELSR1 is found to be causal for non-syndromic, lower extremity lymphedema in a family across three generations. Near-infrared fluorescence lymphatic imaging shows that instead of being propelled within the lumen of well-defined lymphatic vessels, lymph moved in regions of both legs in an unusual fashion and within sheet-like structures.CONCLUSION:CELSRI may be responsible for primary, non-syndromic lymphedema in humans.
