Cell Therapy for Critical Limb Ischemia: Advantages, Limitations, and New Perspectives for Treatment of Patients with Critical Diabetic Vasculopathy

Abstract Purpose of Review To provide a highlight of the current state of cell therapy for the treatment of critical limb ischemia in patients with diabetes. Recent Findings The global incidence of diabetes is constantly growing with consequent challenges for healthcare systems worldwide. In the UK only, NHS costs attributed to diabetic complications, such as peripheral vascular disease, amputation, blindness, renal failure, and stroke, average £10 billion each year, with cost pressure being estimated to get worse. Although giant leaps forward have been registered in the scope of early diagnosis and optimal glycaemic control, an effective treatment for critical limb ischemia is still lacking. The present review aims to provide an update of the ongoing work in the field of regenerative medicine. Recent advancements but also limitations imposed by diabetes on the potential of the approach are addressed. In particular, the review focuses on the perturbation of non-coding RNA networks in progenitor cells and the possibility of using emerging knowledge on molecular mechanisms to design refined protocols for personalized therapy. Summary The field of cell therapy showed rapid progress but has limitations. Significant advances are foreseen in the upcoming years thanks to a better understanding of molecular bottlenecks associated with the metabolic disorders

Recent advances in KEAP1/Nrf2-targeting strategies by phytochemical antioxidants, nanoparticles, and biocompatible scaffolds for the treatment of diabetic cardiovascular complications

Abstract Significance: Modulation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated antioxidant response is a key aspect in the onset of diabetes-related cardiovascular complications. With this review, we provide an overview of the recent advances made in the development of Nrf2-targeting strategies for the treatment of diabetes, with particular attention toward the activation of Nrf2 by natural antioxidant compounds, nanoparticles, and oxidative stress-modulating biocompatible scaffolds. Recent Advances: In the past 30 years, studies addressing the use of antioxidant therapies to treat diabetes have grown exponentially, showing promising but yet inconclusive results. Animal studies and clinical trials on the Nrf2 pathway have shown promising results, suggesting that its activation can delay or reverse some of the cardiovascular impairments in diabetes. Critical Issues: Hyperglycemia- and oscillating glucose levels-induced reactive oxygen species (ROS) accumulation is progressively emerging as a central factor in the onset and progression of diabetes-related cardiovascular complications, including endothelial dysfunction, retinopathy, heart failure, stroke, critical limb ischemia, ulcers, and delayed wound healing. In this context, accumulating evidence suggests a central role for Nrf2-mediated antioxidant response, one of the most studied cellular defensive mechanisms against ROS accumulation. Future Directions: Innovative approaches such as tissue engineering and nanotechnology are converging toward targeting oxidative stress in diabetes. Antioxid. Redox Signal. 36, 707–72

High-level expression of a recombinant active microbial transglutaminase in Escherichia coli

Background: Bacterial transglutaminases are increasingly required as industrial reagents for in vitro modification of proteins in different fields such as in food processing as well as for enzymatic site-specific covalent conjugation of therapeutic proteins to polyethylene glycol to get derivatives with improved clinical performances. In this work we studied the production in Escherichia coli of a recombinant transglutaminase from Streptomyces mobaraensis (microbial transglutaminase or MTGase) as enzymatically active chimeric forms using different expression systems under the control of both lac promoter or thermoinducible phage lambda promoter. Results: Thermoinducible and constitutive expression vectors were constructed expressing Met-MTGase with chimeric LacZ 1-8 PNP 1–20 or LacZ 1–8 fusion protein under different promoters. After transformed in competent Escherichia coli K12 strains were fermented in batch and fed-bach mode in different mediums in order to select the best conditions of expression. The two most performing fusion protein systems namely short thermoinducible LacZ 1–8 Met-MTGase from NP668/1 and long constitutive LacZ 1-8 PNP 1–20 Met-MTGase from NP650/1 has been chosen to compare both efficiency of expression and biochemical qualities of the product. Proteins were extracted, purified to homogeneity and verified as a single peak obtained in RP-HPLC. The LacZ 1-8 PNP 1–20 Met-MTGase fusion protein purified from NP650/1 exhibited an activity of 15 U/mg compared to 24 U/mg for the shorter fusion protein purified from NP668/1 cell strain. Conclusions: Combining the experimental data on expression levels and specific activities of purified MTGase fusion proteins, the chimeric LacZ 1-8 Met-MTGase, which displays an enzymatic activity comparable to the wild-type enzyme, was selected as a candidate for producing microbial transglutaminase for industrial applications.Pubblicat

Increased Aortic Calpain-1 Activity Mediates Age-Associated Angiotensin II Signaling of Vascular Smooth Muscle Cells

Angiotensin II (Ang II) signaling, including matrix metalloproteinase type II (MMP2) activation, has been linked to an age-associated increase in migration capacity of vascular smooth muscle cells (VSMC), and to other proinflammatory features of arterial aging. Calpain-1 activation is required for MMP2 expression in fibroblasts and is induced in cardiomyocytes by Ang II. The consequences of engagement of calpain-1 with its substrates, however, in governing the age-associated proinflammatory status within the arterial wall, remains unknown.The present findings demonstrate that transcription, translation, and activity of calpain-1 are significantly up-regulated in rat aortae or early-passage aortic VSMC from old (30-mo) rats compared to young (8-mo). Dual immunolabeling of the arterial wall indicates that colocalization of calpain-1 and Ang II increases within the aged arterial wall. To further explore the relationship of calpain-1 to Ang II, we chronically infused Ang II into young rats, and treated cultured aortic rings or VSMC with Ang II. We also constructed adenoviruses harboring calpain-1 (CANP1) or its endogenous inhibitor calpastatin (CAST) and infected these into VSMC. Ang II induces calpain-1 expression in the aortic walls in vivo and ex vivo and VSMC in vitro. The Ang II mediated, age-associated increased MMP2 activity and migration in VSMC are both blocked by calpain inhibitor 1 or CAST. Over-expression of calpain-1 in young VSMC results in cleavage of intact vimentin, and an increased migratory capacity mimicking that of old VSMC, which is blocked by the MMP inhibitor, GM6001.Calpain-1 activation is a pivotal molecular event in the age-associated arterial Ang II/MMP2 signaling cascade that is linked to cytoskeleton protein restructuring, and VSMC migration. Therefore, targeting calpain-1 has the potential to delay or reverse the arterial remodeling that underlies age-associated diseases i.e. atherosclerosis

The genetics of exceptional longevity identifies new druggable targets forvascular protection and repair

Therapeutic angiogenesis is a relatively new medical strategy in the field of cardiovascular diseases. The underpinning concept is that angiogenic growth factors or proangiogenic cells could be exploited therapeutically in cardiovascular patients to enhance native revascularization responses to an ischemic insult, thereby accelerating tissue healing. The initial enthusiasm generated by preclinical studies has been tempered by the modest success of clinical trials assessing therapeutic angiogenesis. Similarly, proangiogenic cell therapy has so far not maintained the original promises. Intriguingly, the current trend is to consider regeneration as a prerogative of the youngest organism. Consequentially, the embryonic and foetal models are attracting much attention for clinical translation into corrective modalities in the adulthood. Scientists seem to undervalue the lesson from Mother Nature, e.g. all humans are born young but very few achieve the goal of an exceptional healthy longevity. Either natural experimentation is driven by a supreme intelligence or stochastic phenomena, one has to accept the evidence that healthy longevity is the fruit of an evolutionary process lasting million years. It is therefore extremely likely that results of this natural experimentation are more reliable and translatable than the intensive, but very short human investigation on mechanisms governing repair and regeneration. With this preamble in mind, here we propose to shift the focus from the very beginning to the very end of human life and thus capture the secret of prolonged health span to improve well-being in the adulthood