Eugen Bogdan Petcu

Bisphosphonate-related osteonecrosis of jaw (BRONJ): diagnostic criteria and possible pathogenic mechanisms of an unexpected anti-angiogenic side effect.

Recently, bisphosphonates (BPs) have been widely used in medical practice as anti-resorptive agents owing to their anti-osteoclatic action. In addition, these compounds are also used for their analgesic action and their potential anti-tumour effect. Patients treated with BPs may subsequently develop osteonecrosis of the jaw or maxillary bone after minor local trauma including dental work, recently labelled as bisphosphonate osteonecrosis of jaw (BRONJ). However, the etiopathogenic mechanisms of this pathological condition are poorly understood. Although, several pathways have been proposed for BRONJ occurrence, no single model can explain all morphological changes observed at the macro- and microscopic level. Recent research suggests that BPs may promote an anti-angiogenic effect which contributes directly to the clinical features associated with BRONJ. Remarkably, the anti-angiogenic effect promoting BRONJ might be in keeping with the anti-neoplastic action of BPs. The current review, presents clinical diagnostic criteria. In addition, based on our own experience we describe the histopathological criteria for diagnosis of BRONJ and the possible pathways which may lead to this frustrating pathological condition.

Cellular and Molecular Events Underlying the Dysregulated Response of the Aged Brain to Stroke : A Mini-Review

Background: Age-related brain injuries, including stroke, are a major cause of physical and mental disabilities. Objective: Therefore, studying the basic mechanism underlying functional recovery after brain stroke in aged subjects is of considerable clinical interest. Methods: This review summarizes the effects of age on recovery after stroke in an animal model, with emphasis on the underlying cellular mechanisms. Results: Data from our laboratory and elsewhere indicate that, behaviorally, aged rats were more severely impaired by stroke than young rats, and they also showed diminished functional recovery. Infarct volume did not differ significantly between young and aged animals, but critical differences were apparent in the cytological response to stroke, most notably an age-related acceleration in the development of the glial scar. Early infarct in older rats is associated with premature accumulation of BrdU-positive microglia and astrocytes, persistence of activated oligodendrocytes, a high incidence of neuronal degeneration and accelerated apoptosis. In aged rats, neuroepithelial-positive cells were rapidly incorporated into the glial scar, but these neuroepithelial-like cells did not make a significant contribution to neurogenesis in the infarcted cortex in young or aged animals. The response of plasticity-associated proteins like MAP1B, was delayed in aged rats. Tissue recovery was further delayed by an age-related increase in the amount of the neurotoxic C-terminal fragment of the β-amyloid precursor protein (A-β) at 2 weeks poststroke. Conclusion: The available evidence indicates that the aged brain has the capability to mount a cytoproliferative response to injury, but the timing of the cellular and genetic response to cerebral insult is dysregulated in aged animals, thereby further compromising functional recovery. Elucidating the molecular basis for this phenomenon in the aging brain could yield novel approaches to neurorestoration in the elderly.

In vitro pre-vascularisation of tissue-engineered constructs A co-culture perspective

In vitro pre-vascularization is one of the main vascularization strategies in the tissue engineering field. Culturing cells within a tissue-engineered construct (TEC) prior to implantation provides researchers with a greater degree of control over the fate of the cells. However, balancing the diverse range of different cell culture parameters in vitro is seldom easy and in most cases, especially in highly vascularized tissues, more than one cell type will reside within the cell culture system. Culturing multiple cell types in the same construct presents its own unique challenges and pitfalls. The following review examines endothelial-driven vascularization and evaluates the direct and indirect role other cell types have in vessel and capillary formation. The article then analyses the different parameters researchers can modulate in a co-culture system in order to design optimal tissue-engineered constructs to match desired clinical applications.

Mild systemic inflammation has a neuroprotective effect after stroke in rats

Stroke is accompanied by a strong inflammatory reaction in the brain. Periodontal disease is a chronic local infection which causes a systemic low grade inflammation. We hypothesized that a mild systemic inflammatory reaction as caused by periodontal disease prior to stroke onset, may exert a neuroprotective effect in a rat model of focal ischemia. To test this hypothesis, marginal periodontitis was induced by ligatures on the second maxillary molars in BB/LL Wistar rats for 3 weeks. Two weeks after periodontitis initiation, focal cerebral ischemia was produced by reversible occlusion of the right middle cerebral artery. After a survival time of 7 days after ischemia, rats were killed and bone loss was determined on the buccal and palatinal surfaces of the defleshed jaw. In addition, markers of systemic inflammation were determined in a different group of laboratory animals at 14 days after the onset of periodontitis. The infarct size and markers of the inflammatory reaction in the brain were determined by immunohistochemistry. We found: (i) rats with ligatures exhibited significantly more periodontal bone loss than the control rats; (ii) the development of periodontitis was associated with an elevated gene expression for several markers of systemic inflammation (interleukin-10, transforming growth factor beta 1, tumor necrosis factor alpha, interleukin-1beta and interferon gamma; (iii) rats with periodontitis and a mild systemic inflammation had a significantly reduced infarct volume and a significant reduction in the number of brain macrophages in the infarcted area. In conclusion we found that mild systemic inflammation elicited prior to stroke onset may have a neuroprotective effect in rats by reducing the infarct volume and tissue destruction by brain macrophages.

Standardization of Negative Controls in Diagnostic Immunohistochemistry: Recommendations From the International Ad Hoc Expert Panel

Standardization of controls, both positive and negative controls, is needed for diagnostic immunohistochemistry (dIHC). The use of IHC-negative controls, irrespective of type, although well established, is not standardized. As such, the relevance and applicability of negative controls continues to challenge both pathologists and laboratory budgets. Despite the clear theoretical notion that appropriate controls serve to demonstrate the sensitivity and specificity of the dIHC test, it remains unclear which types of positive and negative controls are applicable and/or useful in day-to-day clinical practice. There is a perceived need to provide “best practice recommendations” for the use of negative controls. This perception is driven not only by logistics and cost issues, but also by increased pressure for accurate IHC testing, especially when IHC is performed for predictive markers, the number of which is rising as personalized medicine continues to develop. Herein, an international ad hoc expert panel reviews classification of negative controls relevant to clinical practice, proposes standard terminology for negative controls, considers the total evidence of IHC specificity that is available to pathologists, and develops a set of recommendations for the use of negative controls in dIHC based on “fit-for-use” principles.

Pathophysiology of the Vascular Wall and its Relevance for Cerebrovascular Disorders in Aged Rodents

Chronic hypertension and cerebral amyloid angiopathy (CAA) are the main pathologies which can induce the rupture of cerebral vessels and intracerebral hemorrhages, as a result of degenerative changes in the vascular wall. A lot of progress has been made in this direction since the successful creation of the first mouse model for the study of Alzheimers disease (AD), as the spectrum of AD pathology includes a plethora of changes found in pure cerebrovascular diseases. We describe here some of these mouse models having important vascular changes that parallel human AD pathology, and more importantly, we show how these models have helped us understand more about the mechanisms that lead to CAA formation. An important cellular event associated with reduced structural and functional recovery after stroke in aged animals is the early formation of a scar in the infarcted region that impairs subsequent neural recovery and repair. We review recent evidence showing that the rapid formation of the glial scar following stroke in aged rats is associated with premature cellular proliferation that originates primarily from the walls of capillaries in the corpus callosum adjacent to the infarcted region. After stroke several vascular mechanisms are turned-on immediately to protect the brain from further damage and help subsequent neuroregeneration and functional recovery. Although does occur after stroke, vasculogenesis is overshadowed in its protective/restorative role by the angiogenesis and arteriogenesis. Understanding the basic mechanisms underlying functional recovery after cerebral stroke in aging subjects is likely to yield new insights into the treatment of brain injury in the clinic.

Targeting p35/Cdk5 Signalling via CIP-Peptide Promotes Angiogenesis in Hypoxia

Cyclin-dependent kinase-5 (Cdk5) is over-expressed in both neurons and microvessels in hypoxic regions of stroke tissue and has a significant pathological role following hyper-phosphorylation leading to calpain-induced cell death. Here, we have identified a critical role of Cdk5 in cytoskeleton/focal dynamics, wherein its activator, p35, redistributes along actin microfilaments of spreading cells co-localising with p(Tyr15)Cdk5, talin/integrin beta-1 at the lamellipodia in polarising cells. Cdk5 inhibition (roscovitine) resulted in actin-cytoskeleton disorganisation, prevention of protein co-localization and inhibition of movement. Cells expressing Cdk5 (D144N) kinase mutant, were unable to spread, migrate and form tube-like structures or sprouts, while Cdk5 wild-type over-expression showed enhanced motility and angiogenesis in vitro, which was maintained during hypoxia. Gene microarray studies demonstrated myocyte enhancer factor (MEF2C) as a substrate for Cdk5-mediated angiogenesis in vitro. MEF2C showed nuclear co-immunoprecipitation with Cdk5 and almost complete inhibition of differentiation and sprout formation following siRNA knock-down. In hypoxia, insertion of Cdk5/p25-inhibitory peptide (CIP) vector preserved and enhanced in vitro angiogenesis. These results demonstrate the existence of critical and complementary signalling pathways through Cdk5 and p35, and through which coordination is a required factor for successful angiogenesis in sustained hypoxic condition.

Citicoline induces angiogenesis improving survival of vascular/human brain microvessel endothelial cells through pathways involving ERK1/2 and insulin receptor substrate-1

BackgroundCiticoline is one of the neuroprotective agents that have been used as a therapy in stroke patients. There is limited published data describing the mechanisms through which it acts.MethodsWe used in vitro angiogenesis assays: migration, proliferation, differentiation into tube-like structures in Matrigel™ and spheroid development assays in human brain microvessel endothelial cells (hCMEC/D3). Western blotting was performed on protein extraction from hCMEC/D3 stimulated with citicoline. An analysis of citicoline signalling pathways was previously studied using a Kinexus phospho-protein screening array. A staurosporin/calcium ionophore-induced apoptosis assay was performed by seeding hCMEC/D3 on to glass coverslips in serum poor medium. In a pilot in vivo study, transient MCAO in rats was carried out with and without citicoline treatment (1000 mg/Kg) applied at the time of occlusion and subsequently every 3 days until euthanasia (21 days). Vascularity of the stroke-affected regions was examined by immunohistochemistry.ResultsCiticoline presented no mitogenic and chemotactic effects on hCMEC/D3; however, it significantly increased wound recovery, the formation of tube-like structures in Matrigel™ and enhanced spheroid development and sprouting. Citicoline induced the expression of phospho-extracellular-signal regulated kinase (ERK)-1/2. Kinexus assays showed an over-expression of insulin receptor substrate-1 (IRS-1). Knock-down of IRS-1 with targeted siRNA in our hCMEC/D3 inhibited the pro-angiogenic effects of citicoline. The percentage of surviving cells was higher in the presence of citicoline. Citicoline treatment significantly increased the numbers of new, active CD105-positive microvessels following MCAO.ConclusionsThe findings demonstrate both a pro-angiogenic and protective effect of citicoline on hCMEC/D3 in vitro and following middle cerebral artery occlusion (MCAO) in vivo.

Bisphosphonate-related osteonecrosis of jaw (BRONJ): an anti-angiogenic side-effect?

Bisphosphonates are recommended in the treatment of osteoporosis and some cancers, in which case they prevent the appearance of bone metastasis. The patients taking bisphosphonates are at increased risk of developing bisphosphonate-related osteonecrosis of jaw (BRONJ) which is characterised by the presence of an un-healing wound after dental surgery. BRONJ might represent an anti-angiogenic side effect. However, the real number of patients with BRONJ might be higher than currently recorded. Considering the differential diagnosis which includes various primary and secondary cancers, a correct histopathological diagnosis is very important. The morphological criteria for diagnosis of BRONJ are highlighted in this material.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1813972972323288

Myeloma-Induced Alloreactive T Cells Arising in Myeloma-Infiltrated Bones Include Double-Positive CD8+CD4+ T Cells: Evidence from Myeloma-Bearing Mouse Model

The graft-versus-myeloma (GVM) effect represents a powerful form of immune attack exerted by alloreactive T cells against multiple myeloma cells, which leads to clinical responses in multiple myeloma transplant recipients. Whether myeloma cells are themselves able to induce alloreactive T cells capable of the GVM effect is not defined. Using adoptive transfer of T naive cells into myeloma-bearing mice (established by transplantation of human RPMI8226-TGL myeloma cells into CD122+ cell-depleted NOD/SCID hosts), we found that myeloma cells induced alloreactive T cells that suppressed myeloma growth and prolonged survival of T cell recipients. Myeloma-induced alloreactive T cells arising in the myeloma-infiltrated bones exerted cytotoxic activity against resident myeloma cells, but limited activity against control myeloma cells obtained from myeloma-bearing mice that did not receive T naive cells. These myeloma-induced alloreactive T cells were derived through multiple CD8+ T cell divisions and enriched in double-positive (DP) T cells coexpressing the CD8αα and CD4 coreceptors. MHC class I expression on myeloma cells and contact with T cells were required for CD8+ T cell divisions and DP-T cell development. DP-T cells present in myeloma-infiltrated bones contained a higher proportion of cells expressing cytotoxic mediators IFN-γ and/or perforin compared with single-positive CD8+ T cells, acquired the capacity to degranulate as measured by CD107 expression, and contributed to an elevated perforin level seen in the myeloma-infiltrated bones. These observations suggest that myeloma-induced alloreactive T cells arising in myeloma-infiltrated bones are enriched with DP-T cells equipped with cytotoxic effector functions that are likely to be involved in the GVM effect.