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].

Nordic Immunohistochemical Quality Control (NordiQC) performs proficiency testing for about 600 pathology laboratories in more than 50 countries. All general results are published on http://www.nordiqc.org. Over-all, about 30% of the staining results on circulated slides from tissue micro arrays have been assessed as insufficient for diagnostic use by an expert group. 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.

BackgroundAdipose-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.MethodsHuman 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.ResultsOur 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.ConclusionsOur 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.

Chronic venous disease (CVD) has a range of clinical presentations, including tortuous, distended veins in lower extremities, increasing skin pigmentation, and in severe cases ulceration of the affected skin. Venous insufficiency, a precursor to CVD characterized by improper return of blood from the lower extremities to the heart, must be studied in its earliest stages at a time when preventative measures could be applied in man. This underscores the need for basic research into biomarkers and genetic predisposing factors affecting the progression of venous disease. Investigation over the past decade has yielded insight into these specific genetic, cellular and molecular mechanisms underlying the development of venous disease. Among the many advances include the elucidation of an increasing role for matrix metalloproteinases as important mediators of the degenerative process involved with venous insufficiency. This may be preceded by an inflammatory process which further contributes to venular degeneration and endothelial dysfunction seen in advanced presentation of disease. Furthermore, genomic analyses have shed light upon temporal expression patterns of matrix remodeling proteins in diseased tissue samples. In this review we examine some of the current findings surrounding cellular, molecular and genetic advances in delineating the etiology of chronic venous disease.

A meeting report for Vascular Biology 2014, held in Monterey, California and organized by the North American Vascular Biology Organization (NAVBO).

BackgroundAngiogenesis is an essential process during tumor development and growth. The murine dorsal skinfold window chamber model has been used for the study of both tumor microvasculature and other vascular diseases, including the study of anti-angiogenic agents in cancer therapy. Hyperspectral imaging of oxygen status of the microvasculature has not been widely used to evaluate response to inhibition of angiogenesis in early tumor cell induced vascular development. This study demonstrates the use of two different classes of anti-angiogenic agents, one targeting the Vascular Endothelial Growth Factor (VEGF) pathway involved with vessel sprouting and the other targeting the Angiopoietin/Tie2 pathway involved in vascular destabilization. Studies evaluated the tumor microvascular response to anti-angiogenic inhibitors in the highly angiogenic renal cell carcinoma induced angiogenesis model.MethodsHuman renal cell carcinoma, Caki-2 cells, were implanted in the murine skinfold window chamber. Mice were treated with either VEGF pathway targeted small molecule inhibitor Sunitinib (100 mg/kg) or with an anti-Ang-2 monoclonal antibody (10 mg/kg) beginning the day of window chamber surgery and tumor cell implantation. Hyperspectral imaging of hemoglobin saturation was used to evaluate both the development and oxygenation of the tumor microvasculature. Tumor volume over time was also assessed over an 11-day period post surgery.ResultsThe window chamber model was useful to demonstrate the inhibition of angiogenesis using the VEGF pathway targeted agent Sunitinib. Results show impairment of tumor microvascular development, reduced oxygenation of tumor-associated vasculature and impairment of tumor volume growth compared to control. On the other hand, this model failed to demonstrate the anti-angiogenic effect of the Ang-2 targeted agent. Follow up experiments suggest that the initial surgery of the window chamber model may interfere with such an agent thus skewing the actual effects on angiogenesis.ConclusionsResults show that this model has great potential to evaluate anti-VEGF, or comparable, targeted agents; however the mere protocol of the window chamber model interferes with proper evaluation of Ang-2 targeted agents. The limitations of this in vivo model in evaluating the response of tumor vasculature to anti-angiogenic agents are discussed.

BackgroundThe understanding of endothelial cell biology has been facilitated by the availability of primary endothelial cell cultures from a variety of sites and species; however, the isolation and maintenance of primary mouse aortic endothelial cells (MAECs) remain a formidable challenge. Culturing MAECs is difficult as they are prone to phenotypic drift during culture. Therefore, there is a need to have a dependable in vitro culture system, wherein the primary endothelial cells retain their properties and phenotypes.MethodsHere, we developed an effective method to prepare immortalized MAEC (iMAEC) lines. Primary MAECs, initially isolated from aortic explants, were immortalized using a retrovirus expressing polyoma middle T-antigen. Immortalized cells were then incubated with DiI-acetylated-low density lipoprotein and sorted via flow cytometry to isolate iMAECs.ResultsiMAECs expressed common markers of endothelial cells, including PECAM1, eNOS, VE-cadherin, and von Willebrand Factor. iMAECs aligned in the direction of imposed laminar shear and retained the ability to form tubes. Using this method, we have generated iMAEC lines from wild-type and various genetically modified mice such as p47phox-/-, eNOS-/-, and caveolin-1-/-.ConclusionIn summary, generation of iMAEC lines from various genetically modified mouse lines provides an invaluable tool to study vascular biology and pathophysiology.

BackgroundThe overexpression of angiopoietin-2 (Ang-2) has both pro-tumorigenic and anti-tumorigenic effects. However, the mechanisms of this protein’s dual effects are poorly understood, and it remains unclear how Ang-2 cooperates with vascular endothelial growth factor (VEGF). In the current study, we investigated the effects of Ang-2 overexpression on nasopharyngeal carcinoma growth in the presence of different levels of VEGF.MethodsAng-2 was introduced into the CNE2 cell line by liposome transfection, and the expression of endogenous VEGF was inhibited by microRNA-mediated RNA interference. CNE2 cells expressing varying levels of Ang-2 and VEGF were injected subcutaneously into the flanks of nude mice. Tumor growth was measured, and vessels from the harvested tumors were analyzed.ResultsThe overexpression of Ang-2 had no obvious effect on CNE2 tumor growth in the presence of endogenous VEGF but significantly inhibited CNE2 tumor growth when the expression of endogenous VEGF was silenced, and the Ang-2/VEGF ratio is negatively correlated with tumor growth. Ang-2 overexpression decreased the percentage of α-SMA-positive cells around the tumor vessels but reduced the microvessel density only in the absence of VEGF.ConclusionsOur results indicate that the effects of Ang-2 on nasopharyngeal carcinoma are highly dependent on the level of VEGF expression, Ang-2/VEGF ratio may offer a novel therapeutic approach for treating human cancer.

Angiogenesis is regulated, under both physiological and pathological conditions, by numerous “non-classic” pro-angiogenic factors, including fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), and placental growth factor (PlGF), and “non-classic” pro-angiogenic factors, including granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), and erythropoietin (EPO). In the context of the most important discoveries in this field, this review article summarizes the important role played by the Italian scientists in the course of the last twenty years.

Polycystic ovarian syndrome (PCOS) ovaries are characterized by increased angiogenesis and hypervascularity. While angiopoietin-1 (Ang-1) and its antagonist, angiopoietin-2 (Ang-2), are essential for ovarian function and angiogenesis, the levels of Ang-1 and Ang-2 in PCOS are unknown. This was a prospective cohort study of 14 PCOS women and 14 matched controls undergoing controlled ovarian stimulation (COS). Serum was collected on day 3, hCG and retrieval days. Follicular fluid (FF) was collected on retrieval day. Serum Ang-1 and Ang-2 levels were constant throughout COS, but serum Ang-1 levels were increased at all time points in PCOS women compared with controls (p < 0.05). No differences between groups were found in serum Ang-2 levels or FF Ang-1 levels. However, FF Ang-2 levels were increased almost 2-fold in PCOS women compared with controls (p < 0.01), and correlated positively with number of oocytes retrieved (r = 0.65, p < 0.0001). This study is the first to provide evidence of an alteration in the Ang-1/Ang-2 system in PCOS women. The biological role of Ang-2 in promoting capillary leakage, the increased Ang-2 FF level in PCOS, and its correlation with number of oocytes suggest that Ang-2 may play an important role in the increased risk of ovarian hyperstimulation in PCOS.