Unraveling the differential mechanisms of revascularization promoted by MSCs & ECFCs from adipose tissue or umbilical cord in a murine model of critical limb-threatening ischemia.

BACKGROUND: Critical limb-threatening ischemia (CLTI) constitutes the most severe manifestation of peripheral artery disease, usually induced by atherosclerosis. CLTI patients suffer from high risk of amputation of the lower extremities and elevated mortality rates, while they have low options for surgical revascularization due to associated comorbidities. Alternatively, cell-based therapeutic strategies represent an effective and safe approach to promote revascularization. However, the variability seen in several factors such as cell combinations or doses applied, have limited their success in clinical trials, being necessary to reach a consensus regarding the optimal "cellular-cocktail" prior further application into the clinic. To achieve so, it is essential to understand the mechanisms by which these cells exert their regenerative properties. Herein, we have evaluated, for the first time, the regenerative and vasculogenic potential of a combination of endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) isolated from adipose-tissue (AT), compared with ECFCs from umbilical cord blood (CB-ECFCs) and AT-MSCs, in a murine model of CLTI. METHODS: Balb-c nude mice (n:32) were distributed in four different groups (n:8/group): control shams, and ischemic mice (after femoral ligation) that received 50 µl of physiological serum alone or a cellular combination of AT-MSCs with either CB-ECFCs or AT-ECFCs. Follow-up of blood flow reperfusion and ischemic symptoms was carried out for 21 days, when mice were sacrificed to evaluate vascular density formation. Moreover, the long-term molecular changes in response to CLTI and both cell combinations were analyzed in a proteomic quantitative approach. RESULTS: AT-MSCs with either AT- or CB-ECFCs, promoted a significant recovery of blood flow in CLTI mice 21 days post-ischemia. Besides, they modulated the inflammatory and necrotic related processes, although the CB group presented the slowest ischemic progression along the assay. Moreover, many proteins involved in the repairing mechanisms promoted by cell treatments were identified. CONCLUSIONS: The combination of AT-MSCs with AT-ECFCs or with CB-ECFCs promoted similar revascularization in CLTI mice, by restoring blood flow levels, together with the modulation of the inflammatory and necrotic processes, and reduction of muscle damage. The protein changes identified are representative of the molecular mechanisms involved in ECFCs and MSCs-induced revascularization (immune response, vascular repair, muscle regeneration, etc.).

Generation of Highly Antioxidant Submicron Particles from Myrtus communis Leaf Extract by Supercritical Antisolvent Extraction Process.

Submicron particles have been produced from an ethanolic extract of Myrtus communnis leaves using supercritical carbon dioxide technology, hereinafter referred to as Supercritical Antisolvent Extraction (SAE). The influence of pressure (9-20 MPa), temperature (308 and 328 K) and injection rate (3 and 8 mL/min) on the particles' precipitation has been investigated, and it has been confirmed that increases in pressure and temperature led to smaller particle sizes. The obtained particles had a quasi-spherical shape with sizes ranging from 0.42 to 1.32 µm. Moreover, the bioactivity of the generated particles was assessed and large contents of phenolic compounds with a high antioxidant activity were measured. The particles were also subjected to in vitro studies against oxidative stress. The myrtle particles demonstrated cytoprotective properties when applied at low concentrations (1 µM) to macrophage cell lines.

Nrf2 and Heme Oxygenase-1 Involvement in Atherosclerosis Related Oxidative Stress.

Atherosclerosis remains the underlying process responsible for cardiovascular diseases and the high mortality rates associated. This chronic inflammatory disease progresses with the formation of occlusive atherosclerotic plaques over the inner walls of vascular vessels, with oxidative stress being an important element of this pathology. Oxidation of low-density lipoproteins (ox-LDL) induces endothelial dysfunction, foam cell activation, and inflammatory response, resulting in the formation of fatty streaks in the atherosclerotic wall. With this in mind, different approaches aim to reduce oxidative damage as a strategy to tackle the progression of atherosclerosis. Special attention has been paid in recent years to the transcription factor Nrf2 and its downstream-regulated protein heme oxygenase-1 (HO-1), both known to provide protection against atherosclerotic injury. In the current review, we summarize the involvement of oxidative stress in atherosclerosis, focusing on the role that these antioxidant molecules exert, as well as the potential therapeutic strategies applied to enhance their antioxidant and antiatherogenic properties.

N-glycosylation profile analysis of Trastuzumab biosimilar candidates by Normal Phase Liquid Chromatography and MALDI-TOF MS approaches.

The pharmaceutical market has entered an era in which the production of new therapeutics is being often replaced by "biosimilars", copies of already commercialized products waiting for the patents to expire in order to be distributed in a more competitive and affordable manners. Due to its relevance, the ErbB2-targeted monoclonal antibody Trastuzumab (Herceptin) used as breast cancer therapy is one of the main targets in the production of biosimilars. A major challenge is to produce antibodies with the same or the closest N-glycosylation pattern seen in the commercialized drug. Several factors, such as growing conditions or cell types employed, can determine the final composition and structure of the glycans, significantly affecting the properties of the generated antibodies. Therefore, an appropriate characterization is essential. In the present study, we describe two different but complementary strategies to characterize the N-glycosylation of two biosimilar candidates of Trastuzumab. In the first case, N-glycans are fluorescently labeled and separated by Normal Phase HPLC. Different sugars will elute at different times and can be identified using specific oligosaccharide standards. In the second approach, released glycans are permethylated and analyzed by MALDI-TOF MS, being able to determine the structure because of the differential sugar masses. BIOLOGICAL SIGNIFICANCE: The characterization of the N-glycosylation sites of therapeutic recombinant monoclonal antibodies (mAbs) is usually one of the most critical and time consuming steps in the developing process of biosimilars or any other glycosylated drug. Herein we describe two different but complementary approaches to characterize mAbs glycosylation patterns, the use of glycan fluorescence labeling coupled to HPLC and MALDI-TOF MS profile analysis. This article is part of a Special Issue entitled: HUPO 2014.