The Cleavage-Specific Tau 12A12mAb Exerts an Anti-Amyloidogenic Action by Modulating the Endocytic and Bioenergetic Pathways in Alzheimer's Disease Mouse Model.

Beyond deficits in hippocampal-dependent episodic memory, Alzheimer's Disease (AD) features sensory impairment in visual cognition consistent with extensive neuropathology in the retina. 12A12 is a monoclonal cleavage specific antibody (mAb) that in vivo selectively neutralizes the AD-relevant, harmful N-terminal 20-22 kDa tau fragment(s) (i.e., NH2htau) without affecting the full-length normal protein. When systemically injected into the Tg2576 mouse model overexpressing a mutant form of Amyloid Precursor Protein (APP), APPK670/671L linked to early onset familial AD, this conformation-specific tau mAb successfully reduces the NH2htau accumulating both in their brain and retina and, thus, markedly alleviates the phenotype-associated signs. By means of a combined biochemical and metabolic experimental approach, we report that 12A12mAb downregulates the steady state expression levels of APP and Beta-Secretase 1 (BACE-1) and, thus, limits the Amyloid beta (Aß) production both in the hippocampus and retina from this AD animal model. The local, antibody-mediated anti-amyloidogenic action is paralleled in vivo by coordinated modulation of the endocytic (BIN1, RIN3) and bioenergetic (glycolysis and L-Lactate) pathways. These findings indicate for the first time that similar molecular and metabolic retino-cerebral pathways are modulated in a coordinated fashion in response to 12A12mAb treatment to tackle the neurosensorial Aß accumulation in AD neurodegeneration.

Tau Cleavage Contributes to Cognitive Dysfunction in Strepto-Zotocin-Induced Sporadic Alzheimer's Disease (sAD) Mouse Model.

Tau cleavage plays a crucial role in the onset and progression of Alzheimer's Disease (AD), a widespread neurodegenerative disease whose incidence is expected to increase in the next years. While genetic and familial forms of AD (fAD) occurring early in life represent less than 1%, the sporadic and late-onset ones (sAD) are the most common, with ageing being an important risk factor. Intracerebroventricular (ICV) infusion of streptozotocin (STZ)-a compound used in the systemic induction of diabetes due to its ability to damage the pancreatic ß cells and to induce insulin resistance-mimics in rodents several behavioral, molecular and histopathological hallmarks of sAD, including memory/learning disturbance, amyloid-ß (Aß) accumulation, tau hyperphosphorylation, oxidative stress and brain glucose hypometabolism. We have demonstrated that pathological truncation of tau at its N-terminal domain occurs into hippocampi from two well-established transgenic lines of fAD animal models, such as Tg2576 and 3xTg mice, and that it's in vivo neutralization via intravenous (i.v.) administration of the cleavage-specific anti-tau 12A12 monoclonal antibody (mAb) is strongly neuroprotective. Here, we report the therapeutic efficacy of 12A12mAb in STZ-infused mice after 14 days (short-term immunization, STIR) and 21 days (long-term immunization regimen, LTIR) of i.v. delivery. A virtually complete recovery was detected after three weeks of 12A12mAb immunization in both novel object recognition test (NORT) and object place recognition task (OPRT). Consistently, three weeks of this immunization regimen relieved in hippocampi from ICV-STZ mice the AD-like up-regulation of amyloid precursor protein (APP), the tau hyperphosphorylation and neuroinflammation, likely due to modulation of the PI3K/AKT/GSK3-ß axis and the AMP-activated protein kinase (AMPK) activities. Cerebral oxidative stress, mitochondrial impairment, synaptic and histological alterations occurring in STZ-infused mice were also strongly attenuated by 12A12mAb delivery. These results further strengthen the causal role of N-terminal tau cleavage in AD pathogenesis and indicate that its specific neutralization by non-invasive administration of 12A12mAb can be a therapeutic option for both fAD and sAD patients, as well as for those showing type 2 diabetes as a comorbidity.

Optimization of DNA extraction from dental remains.

Efficient DNA extraction procedures is a critical step involved in the process of successful DNA analysis of such samples. Various protocols have been devised for the genomic DNA extraction from human tissues and forensic stains, such as dental tissue that is the skeletal part that better preserves DNA over time. However DNA recovery is low and protocols require labor-intensive and time-consuming step prior to isolating genetic material. Herein, we describe an extremely fast procedure of DNA extraction from teeth compared to classical method. Sixteen teeth of 100-year-old human remains were divided into two groups of 8 teeth and we compared DNA yield, in term of quantity and quality, starting from two different sample preparation steps. Specifically, teeth of group 1 were treated with a classic technique based on several steps of pulverization and decalcification, while teeth of group 2 were processed following a new procedure to withdraw dental pulp. In the next phase, the samples of both group underwent the same procedure of extraction, quantification and DNA profile analysis. Our findings provide an alternative protocol to obtain a higher amount of good quality DNA in a fast time procedure, helpful for forensic and anthropological studies.

Human DDX3 protein is a valuable target to develop broad spectrum antiviral agents.

Targeting a host factor essential for the replication of different viruses but not for the cells offers a higher genetic barrier to the development of resistance, may simplify therapy regimens for coinfections, and facilitates management of emerging viral diseases. DEAD-box polypeptide 3 (DDX3) is a human host factor required for the replication of several DNA and RNA viruses, including some of the most challenging human pathogens currently circulating, such as HIV-1, Hepatitis C virus, Dengue virus, and West Nile virus. Herein, we showed for the first time, to our knowledge, that the inhibition of DDX3 by a small molecule could be successfully exploited for the development of a broad spectrum antiviral agent. In addition to the multiple antiviral activities, hit compound 16d retained full activity against drug-resistant HIV-1 strains in the absence of cellular toxicity. Pharmacokinetics and toxicity studies in rats confirmed a good safety profile and bioavailability of 16d. Thus, DDX3 is here validated as a valuable therapeutic target.

Sexual dimorphism in medulloblastoma features.

AIMS: Male sex is a risk factor for medulloblastoma (MB), and is also a negative predictor for clinical outcome. The aim of this study was to assess sex differences in tumour biological features and hormone receptor profiles in a cohort of MB patients. METHODS AND RESULTS: Sixty-four MBs and five normal cerebella were included in the study. Cell proliferation (Ki67), apoptosis (cleaved caspase-3) and microvessel density (CD31) were evaluated in tumours by immunohistochemistry. Tissues were analysed for oestrogen receptor (ER)α, ERß1, ERß2, ERß5 and androgen receptor (AR) expression. The results demonstrated sex-specific features in MBs, with tumours from females showing a higher apoptosis/proliferation ratio and less tumour vascularization than tumours from males. MBs were negative for ERα and AR, but expressed ERß isoforms at similar levels between the sexes. Altogether, these findings indicate that signalling mechanisms that control cell turnover and angiogenesis operate more efficiently in females than in males. The lack of sex differences in the hormone receptor profiles suggests that circulating oestrogens could be the major determinants of the sexual dimorphism observed in MB features. CONCLUSIONS: Here, we provide molecular support for epidemiological data showing sex differences in MB incidence and outcome, completely defining the hormone receptor profile of the tumours.

Increased FGF23 serum level is associated with unstable carotid plaque in type 2 diabetic subjects with internal carotid stenosis.

BACKGROUND: The object of this study was to investigate the potential role of FGF23 on plaque stability in type 2 diabetic patients with internal carotid artery stenosis. METHODS: In this retrospective observational study, we analyzed FGF23 serum level in 361 type 2 diabetic patients with internal carotid artery stenosis undergoing carotid endarterectomy and in 598 diabetic controls without carotid atherosclerosis. RESULTS: We found that FGF23 median serum levels was significantly higher in patients than in diabetic controls [67.7 (59.5-77.8) pg/mL and 43.89 (37.5-50.4), P < 0.001] and was significantly and independently associated with unstable plaque in patients with internal carotid artery stenosis [OR, 5,71 (95% CI, 2.09-15.29]. CONCLUSIONS: We have found, for the first time, that FGF23 could be associated with unstable plaque in type 2 diabetic patients with internal carotid artery stenosis.

Qualitative and quantitative differences of adipose-derived stromal cells from superficial and deep subcutaneous lipoaspirates: a matter of fat.

BACKGROUND AIMS: Subcutaneous fat represents a valuable reservoir of adipose-derived stem cells (ASCs) in the stromal vascular fraction (SVF), widely exploited in regenerative medicine applications, being easily harvested through lipoaspiration. The lack of standardized procedures for autologous fat grafting guided research efforts aimed at identifying possible differences related to the harvesting site, which may affect cell isolation yield, cell growth properties and clinical outcomes. Subcutaneous fat features a complex architecture: the superficial fascia separates superficial adipose tissue (SAT) from deep layer tissue (DAT). We aimed to unravel the differences between SAT and DAT, considering morphological structure, SVF composition, and ASC properties. METHODS: SAT and DAT were collected from female donors and comparatively analyzed to evaluate cellular yield and viability, morphology, immunophenotype and molecular profile. ASCs were isolated in primary culture and used for in vitro differentiation assays. SAT and DAT from cadaver donors were also analyzed through histology and immunohistochemistry to assess morphology and cell localization within the hypoderm. RESULTS: Liposuctioned SAT contained a higher stromal tissue compound, along with a higher proportion of CD105-positive cells, compared with DAT from the same harvesting site. Also, cells isolated from SAT displayed increased multipotency and stemness features. All differences were mainly evidenced in specimens harvested from the abdominal region. According to our results, SAT features overall increased stem properties. CONCLUSIONS: Given that subcutaneous adipose tissue is currently exploited as the gold standard source for high-yield isolation of adult stem cells, these results may provide precious hints toward the definition of standardized protocols for microharvesting.

Angiogenic impairment of the vascular endothelium: a novel mechanism and potential therapeutic target in muscular dystrophy.

OBJECTIVE: Dystrophin, the missing or defective protein in Duchenne muscular dystrophy, is expressed not only in muscle cells but also in vascular endothelial cells (ECs). In this study, we assessed the effects of dystrophin deficiency on the angiogenic capacities of ECs. APPROACH AND RESULTS: We isolated vascular ECs from mdx mice, the murine equivalent of Duchenne muscular dystrophy in humans, and wild-type controls, and we found that mdx-derived ECs have impaired angiogenic properties, in terms of migration, proliferation, and tube formation. They also undergo increased apoptosis in vitro compared with wild-type cells and have increased senescence-associated ß-galactosidase activity. Mdx-derived ECs also display reduced ability to support myoblast proliferation when cocultured with satellite cell-derived primary myoblasts. These endothelial defects are mirrored by systemic impairment of angiogenesis in vivo, both on induction of ischemia, stimulation with growth factors in the corneal model and matrigel plug assays, and tumor growth. We also found that dystrophin forms a complex with endothelial NO synthase and caveolin-1 in ECs, and that NO production and cGMP formation are compromised in ECs isolated from mdx mice. Interestingly, treatment with aspirin enhances production of both cGMP and NO in dystrophic ECs, whereas low-dose aspirin improves the dystrophic phenotype of mdx mice in vivo, in terms of resistance to physical exercise, muscle fiber permeability, and capillary density. CONCLUSIONS: These findings demonstrate that impaired angiogenesis is a novel player and potential therapeutic target in Duchenne muscular dystrophy.

Anterior video-assisted approach to the craniovertebral junction: transnasal or transoral? A cadaver study.

BACKGROUND: Endoscopy represents both an alternative and useful complement to the standard microsurgical approach to the anterior craniovertebral junction (CVJ). Nevertheless, few studies provide an experimental comparison between transnasal and transoral endoscopic control on CVJ. We compared the surgical exposition angle and the working channel volume of both the transnasal and transoral approaches in the cadaver. METHODS: Eleven fresh non-perfused cadavers were studied. Transnasal and transoral linear and angled exposure of the CVJ were evaluated by means of X-ray and CT scan both in sagittal and lateral planes. RESULTS: The transoral endoscopic surgical exposition was wider compared with the transnasal in anterior and lateral projections:(1)in the sagittal plane, both in vertical exposition (transnasal inferior to transoral from 5.89 % to 76.48 %, average 35.89 %) and in vertical surgical angle (from 22 % to 77.42 %, average 56.53 %); (2)in the coronal plane, both in coronal exposition (transnasal inferior to transoral from 50.77 % to 83.88 %, average 70.34 %) and in coronal surgical angle (from 65.58 % to 86.71 %, average 76.70 %). The sagittal surgical domain was found to spanning from the inferior third of the clivus to C3 with the transoral and from the middle third of the clivus to the nasopalatal line (NPL) with the transnasal approach. The overlapping surgical domain area was found to be the inferior third of the clivus. CONCLUSIONS: The endoscope assisted transoral approach allows a better surgical control of the CVJ. It provides a better CVJ exposure, in sagittal and transverse planes, providing a larger working channel and an easier manoeuvrability. The transnasal approach is limited in caudal direction down to the NPL, otherwise the transoral approach is limited in the rostral direction with a maximum to the foramen magnum in normal specimen. In every individual case, pros and cons of the appropriate approach have to be taken into account as well as the choice of a combined transnasal and transoral approaches strategy.

Contribution of chitinase A's C-terminal vacuolar sorting determinant to the study of soluble protein compartmentation.

Plant chitinases have been studied for their importance in the defense of crop plants from pathogen attacks and for their peculiar vacuolar sorting determinants. A peculiarity of the sequence of many family 19 chitinases is the presence of a C-terminal extension that seems to be important for their correct recognition by the vacuole sorting machinery. The 7 amino acids long C-terminal vacuolar sorting determinant (CtVSD) of tobacco chitinase A is necessary and sufficient for the transport to the vacuole. This VSD shares no homology with other CtVSDs such as the phaseolin's tetrapeptide AFVY (AlaPheValTyr) and it is also sorted by different mechanisms. While a receptor for this signal has not yet been convincingly identified, the research using the chitinase CtVSD has been very informative, leading to the observation of phenomena otherwise difficult to observe such as the presence of separate vacuoles in differentiating cells and the existence of a Golgi-independent route to the vacuole. Thanks to these new insights in the endoplasmic reticulum (ER)-to-vacuole transport, GFPChi (Green Fluorescent Protein carrying the chitinase A CtVSD) and other markers based on chitinase signals will continue to help the investigation of vacuolar biogenesis in plants.

SARS-CoV-2-Related Olfactory Dysfunction: Autopsy Findings, Histopathology, and Evaluation of Viral RNA and ACE2 Expression in Olfactory Bulbs.

BACKGROUND: The COVID-19 pandemic has been a health emergency with a significant impact on the world due to its high infectiousness. The disease, primarily identified in the lower respiratory tract, develops with numerous clinical symptoms affecting multiple organs and displays a clinical finding of anosmia. Several authors have investigated the pathogenetic mechanisms of the olfactory disturbances caused by SARS-CoV-2 infection, proposing different hypotheses and showing contradictory results. Since uncertainties remain about possible virus neurotropism and direct damage to the olfactory bulb, we investigated the expression of SARS-CoV-2 as well as ACE2 receptor transcripts in autoptic lung and olfactory bulb tissues, with respect to the histopathological features. METHODS: Twenty-five COVID-19 olfactory bulbs and lung tissues were randomly collected from 200 initial autopsies performed during the COVID-19 pandemic. Routine diagnosis was based on clinical and radiological findings and were confirmed with post-mortem swabs. Real-time RT-PCR for SARS-CoV-2 and ACE2 receptor RNA was carried out on autoptic FFPE lung and olfactory bulb tissues. Histological staining was performed on tissue specimens and compared with the molecular data. RESULTS: While real-time RT-PCR for SARS-CoV-2 was positive in 23 out of 25 lung samples, the viral RNA expression was absent in olfactory bulbs. ACE2-receptor RNA was present in all tissues examined, being highly expressed in lung samples than olfactory bulbs. CONCLUSIONS: Our finding suggests that COVID-19 anosmia is not only due to neurotropism and the direct action of SARS-CoV-2 entering the olfactory bulb. The mechanism of SARS-CoV-2 neuropathogenesis in the olfactory bulb requires a better elucidation and further research studies to mitigate the olfactory bulb damage associated with virus action.

US-guided application of Nd:YAG laser in porcine pancreatic tissue: an ex vivo study and numerical simulation.

BACKGROUND: Laser ablation (LA) with a neodymium-doped yttrium aluminum garnet (Nd:YAG) laser is a minimally invasive approach able to achieve a high rate of complete tissue necrosis. In a previous study we described the feasibility of EUS-guided Nd:YAG pancreas LA performed in vivo in a porcine model. OBJECTIVE: To establish the best laser setting of Nd:YAG lasers for pancreatic tissue ablation. A secondary aim was to investigate the prediction capability of a mathematical model on ablation volume. DESIGN: Ex vivo animal study. SETTING: Hospital animal laboratory. SUBJECTS: Explanted pancreatic glands from 60 healthy farm pigs. INTERVENTION: Laser output powers (OP) of 1.5, 3, 6, 10, 15, and 20 W were supplied. Ten trials for each OP were performed under US guidance on ex vivo healthy porcine pancreatic tissue. MAIN OUTCOME MEASUREMENTS: Ablation volume (Va) and central carbonization volume (Vc) were measured on histologic specimens as the sum of the lesion areas multiplied by the thickness of each slide. The theoretical model of the laser-tissue interaction was based on the Pennes equation. RESULTS: A circumscribed ablation zone was observed in all histologic specimens. Va values grow with the increase of the OP up to 10 W and reach a plateau between 10 and 20 W. The trend of Vc values rises constantly until 20 W. The theoretical model shows a good agreement with experimental Va and Vc for OP between 1.5 and 10 W. LIMITATIONS: Ex vivo study. CONCLUSION: Volumes recorded suggest that the best laser OP could be the lowest one to obtain similar Va with smaller Vc in order to avoid the risk of thermal injury to the surrounding tissue. The good agreement between the two models demonstrates the prediction capability of the theoretical model on laser-induced ablation volume in an ex vivo animal model and supports its potential use for estimating the ablation size at different laser OPs.

COVID-19 and Alzheimer's Disease Share Common Neurological and Ophthalmological Manifestations: A Bidirectional Risk in the Post-Pandemic Future.

A growing body of evidence indicates that a neuropathological cross-talk takes place between the coronavirus disease 2019 (COVID-19) -the pandemic severe pneumonia that has had a tremendous impact on the global economy and health since three years after its outbreak in December 2019- and Alzheimer's Disease (AD), the leading cause of dementia among human beings, reaching 139 million by the year 2050. Even though COVID-19 is a primary respiratory disease, its causative agent, the so-called Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), is also endowed with high neuro-invasive potential (Neurocovid). The neurological complications of COVID-19, resulting from the direct viral entry into the Central Nervous System (CNS) and/or indirect systemic inflammation and dysregulated activation of immune response, encompass memory decline and anosmia which are typically associated with AD symptomatology. In addition, patients diagnosed with AD are more vulnerable to SARS-CoV-2 infection and are inclined to more severe clinical outcomes. In the present review, we better elucidate the intimate connection between COVID-19 and AD by summarizing the involved risk factors/targets and the underlying biological mechanisms shared by these two disorders with a particular focus on the Angiotensin-Converting Enzyme 2 (ACE2) receptor, APOlipoprotein E (APOE), aging, neuroinflammation and cellular pathways associated with the Amyloid Precursor Protein (APP)/Amyloid beta (Aß) and tau neuropathologies. Finally, the involvement of ophthalmological manifestations, including vitreo-retinal abnormalities and visual deficits, in both COVID-19 and AD are also discussed. Understanding the common physiopathological aspects linking COVID-19 and AD will pave the way to novel management and diagnostic/therapeutic approaches to cope with them in the post-pandemic future.

Exfoliative Cytology and Genetic Analysis for a Non-Invasive Approach to the Diagnosis of White Sponge Nevus: Case Series.

BACKGROUND: White Sponge Nevus (WSN) is a rare benign disorder associated with mutations in genes coding for cytokeratin 4 (KRT4) and 13 (KRT13) characterized by dyskeratotic hyperplasia of mucous membranes. This study was aimed at examining different approaches (cytology, pathology and genetic analysis) to WSN diagnosis. METHODS: A series of four patients with asymptomatic white diffuse oral lesions were evaluated and, before performing an incisional biopsy for pathology, an oral brush Thin Prep was collected for exfoliative liquid-based cytology (LBC). DNA for genetic analysis was also obtained from patients and both their parents, using buccal swabs. RESULTS: Pathology and cytology showed similar results, leading to the same diagnosis of hyperkeratotic epithelium with acanthosis and spongiosis, without atypia, demonstrating the efficiency of LBC for the differential diagnosis. Sequencing analysis revealed at least 6 rare variants in the KRT4 and KRT13 genes in each patient, contributed in part by both unaffected parents. CONCLUSIONS: Thin Prep for oral exfoliative cytology and genetic analysis are sufficient for an accurate diagnosis of WSN. The combination of cytological and genetic analyses could substitute the histologic exam, providing a non-invasive alternative for incisional biopsy.

Dittrichia viscosa Selection Strategy Based on Stress Produces Stable Clonal Lines for Phytoremediation Applications.

Dittrichia viscosa uptake and translocation of the metalloid As is not fully understood and some data are contradictory, but its adaptability to this pollutant is known and is dependent on its genetic variability. D. viscosa is not a hyperaccumulator plant, but it can grow in high-drought conditions while still producing large biomass, even tolerating significant concentrations of As3+ and As5+. In spite of these remarkable characteristics, adaptive modification of performances is not predictable in wild populations. In previous work, we established experimental clonal populations to perform a functional study on the aquaporin NIP1.1. Here, we propose a strategy to select a clonal population of D. viscosa with a defined phenotype related to As tolerance and to reduced NIP1.1 expression levels for phytoremediation applications. From the previous work, we selected four independent clones, two of them belonging to the weak population (W8 and W9) and the other two belonging to the strong population (S1 and S3). The weak and strong populations differ for a different expression ratio root/shoot of DvNip1;1 that brings a different tolerance to As presence. The stress response of the populations, revealed by the CAT enzymatic test, was statistically correlated to the clones, but not to As uptake. Performance of the selected plants on a second unrelated metallic pollutant, Cd, was evaluated, showing that Cd uptake is also independent from the tolerant phenotype. In vitro culture methods using solid media and temporary immersion bioreactors were compared to propose an optimized combined protocol. The procedure yielded propagation of genetically stable tolerant clonal lines with good uptake of As and Cd. The plants, mass-produced with the developed in vitro protocol, were able to maintain their acquired abilities and are potentially able be later applied in phytoremediation or contaminated areas' re-naturalization.

Correction: Anglana et al. Dittrichia viscosa Selection Strategy Based on Stress Produces Stable Clonal Lines for Phytoremediation Applications. Plants 2023, 12, 2499.

In the original publication [...].

Biophysical and proteomic analyses of Pseudomonas syringae pv. tomato DC3000 extracellular vesicles suggest adaptive functions during plant infection.

Vesiculation is a process employed by Gram-negative bacteria to release extracellular vesicles (EVs) into the environment. EVs from pathogenic bacteria play functions in host immune modulation, elimination of host defenses, and acquisition of nutrients from the host. Here, we observed EV production of the bacterial speck disease causal agent, Pseudomonas syringae pv. tomato (Pto) DC3000, as outer membrane vesicle release. Mass spectrometry identified 369 proteins enriched in Pto DC3000 EVs. The EV samples contained known immunomodulatory proteins and could induce plant immune responses mediated by bacterial flagellin. Having identified two biomarkers for EV detection, we provide evidence for Pto DC3000 releasing EVs during plant infection. Bioinformatic analysis of the EV-enriched proteins suggests a role for EVs in antibiotic defense and iron acquisition. Thus, our data provide insights into the strategies this pathogen may use to develop in a plant environment. IMPORTANCE The release of extracellular vesicles (EVs) into the environment is ubiquitous among bacteria. Vesiculation has been recognized as an important mechanism of bacterial pathogenesis and human disease but is poorly understood in phytopathogenic bacteria. Our research addresses the role of bacterial EVs in plant infection. In this work, we show that the causal agent of bacterial speck disease, Pseudomonas syringae pv. tomato, produces EVs during plant infection. Our data suggest that EVs may help the bacteria to adapt to environments, e.g., when iron could be limiting such as the plant apoplast, laying the foundation for studying the factors that phytopathogenic bacteria use to thrive in the plant environment.

Combined therapy with sonic hedgehog gene transfer and bone marrow-derived endothelial progenitor cells enhances angiogenesis and myogenesis in the ischemic skeletal muscle.

We have previously demonstrated that sonic hedgehog (Shh) gene transfer improves angiogenesis in the setting of ischemia by upregulating the expression of multiple growth factors and enhancing the incorporation of endogenous bone marrow (BM)-derived endothelial progenitor cells (EPCs). In this study, we hypothesized that combined therapy with Shh gene transfer and BM-derived EPCs is more effective than Shh gene therapy alone in an experimental model of peripheral limb ischemia. We used old mice, which have a significantly reduced angiogenic response to ischemia, and compared the ability of Shh gene transfer, exogenous EPCs, or both to improve regeneration after ischemia. We found a significantly higher capillary density in the Shh + EPC-treated muscles compared to the other experimental groups. We also found that Shh gene transfer increases the incorporation and survival of transplanted EPCs. Finally, we found a significantly higher number of regenerating myofibers in the ischemic muscles of mice receiving combined treatment with Shh and BM-derived EPCs. In summary, the combination of Shh gene transfer and BM-derived EPCs more effectively promotes angiogenesis and muscle regeneration than each treatment individually and merits further investigation for its potential beneficial effects in ischemic diseases.

Assessment of the genetic effects of polymorphisms in the osteoprotegerin gene, TNFRSF11B, on serum osteoprotegerin levels and carotid plaque vulnerability.

BACKGROUND AND PURPOSE: Osteoprotegerin (OPG) is a secretory glycoprotein which belongs to the tumor necrosis factor receptor family. Various mechanisms have been suggested by which calcification might alter atherosclerotic plaque stability, but the significance of this intimal calcification is controversial. High concentrations of OPG have been associated with the presence of vascular and cardiovascular diseases. This study was designed to assess the association between gene polymorphisms of the OPG gene (TNFRSF11B), the serum OPG level, and plaque stability in patients with carotid atherosclerosis. METHODS: We studied 177 patients with internal carotid artery stenosis who underwent carotid endarterectomy and also 303 controls. Carotid endarterectomy samples removed from patients were assessed by immunohistochemistry. Concentrations of OPG were measured and gene polymorphisms were examined by polymerase chain reaction and restriction enzyme analysis and were compared, initially between patients with carotid atherosclerosis and controls, and subsequently between stable and unstable carotid plaques. RESULTS: We found that the GG genotype of the T245G polymorphism, the CC genotype of the T950C polymorphism, and the CC genotype of the G1181C polymorphism were significantly higher in patients with carotid plaque than in controls (21.5% versus 10.9% , P<0.01; 15.8% versus 7.6%, P<0.01; and 20.3% versus 10.9%, P<0.01, respectively) and that these polymorphisms were associated with high serum OPG levels (4.02 [3.07] versus 2.94 [1.81] pmol/L; P<0.01), which were significantly higher in patients with unstable atherosclerotic plaques (5.86 [4.02] versus 3.53 [1.87] pmol/L; P<0.01). CONCLUSIONS: The TNFRSF11B gene polymorphisms studied are associated with high serum OPG levels and might be potential markers for plaque instability.

Glycaemic variability affects ischaemia-induced angiogenesis in diabetic mice.

The aim of the present study was to investigate the role of GV (glycaemic variability) in diabetic vascular complications and to explore the molecular pathways modulated by glycaemic 'swings'. We developed a murine model. A total of 30 diabetic mice received once daily basal insulin administration plus two oral boluses of glucose solution (GV group, named 'V') and 30 diabetic mice received once daily basal insulin plus two oral boluses of saline solution (stable hyperglycaemia group, named 'S') for a period of 30 days. Glycaemia was measured eight times daily to detect GV. Finally, postischaemic vascularization, induced by hindlimb ischaemia 30 days after diabetes onset, was evaluated. We found that GV was significantly different between S and V groups, whereas no significant difference in the mean glycaemic values was detected. Laser Doppler perfusion imaging and histological analyses revealed that the ischaemia-induced angiogenesis was significantly impaired in V mice compared with S group, after ischaemic injury. In addition, immunostaining and Western blot analyses revealed that impaired angiogenic response in V mice occurred in association with reduced VEGF (vascular endothelial growth factor) production and decreased eNOS (endothelial nitric oxide synthase) and Akt (also called protein kinase B) phosphorylation. In conclusion, we describe a murine model of GV. GV causes an impairment of ischaemia-induced angiogenesis in diabetes, likely to be independent of changes in average blood glucose levels, and this impaired collateral vessel formation is associated with an alteration of the VEGF pathway.