Functional characterisation of a HOX/PBX transcriptional complex

Hox genes control cell fates and specify regional identities during vertebrate development. Hox proteins relaxed DNA-binding selectivity in vitro, suggests that Hox specificity of action is achieved in vivo through the action of transcriptional cofactors. Members of the EXD/PBX family of homeoproteins have been proposed for such a role on the basis of genetic and biochemical evidence. In this work we show that the human Pbxl and HOXBl proteins can cooperatively activate transcription through a genetically charaterized Hox responsive element, the autoregulatory enhancer of the mouse Hoxb-1 gene (b-1ARE), which directs the spatially restricted expression of Hoxb-1 in the fourth rhombomere during hindbrain development. On the b-lARE, only a restricted subset of HOX proteins is able to bind cooperatively and activate transcription. Selective recognition of the b-lARE is mediated by the N-terminal region of the HOX homeodomain. The DNA binding and protein-protein interaction functions of HOXBl and Pbxl are all necessary for the assembly of a transcriptionally active complex on the b-lARE. Functional dissection of the complex allowed the localization of the main activation domain in the HOXBl N-terminal region, and of an additional one in the C-terminal region of Pbxl, which is absent in one of its two alternative splicing isoforms. The transcriptional activity of HOX/Pbxl complexes on the b-lARE element is maximal and further restricted in embryonal carcinoma (EC) cells compared to other cell lines. Mutational analysis shows that an octamer-binding protein consensus site on the b-lARE contributes to the activity of the enhancer in transfected cells and it is bound in vitro by octamer-like proteins from both EC cells and embryonic extracts

Sirtuins 1–7 expression in human adipose-derived stem cells from subcutaneous and visceral fat depots: influence of obesity and hypoxia

The sirtuin family comprises seven NAD+-dependent deacetylases which control the overall health of organisms through the regulation of pleiotropic metabolic pathways. Sirtuins are important modulators of adipose tissue metabolism and their expression is higher in lean than obese subjects. At present, the role of sirtuins in adipose-derived stem cells has not been investigated yet. Therefore, in this study, we evaluated the expression of the complete panel of sirtuins in adipose-derived stem cells isolated from both subcutaneous and visceral fat of non-obese and obese subjects. We aimed at investigating the influence of obesity on sirtuins' levels, their role in obesity-associated inflammation, and the relationship with the peroxisome proliferator-activated receptor delta, which also plays functions in adipose tissue metabolism. The mRNA levels in the four types of adipose-derived stem cells were evaluated by quantitative polymerase chain reaction, in untreated cells and also after 8 h of hypoxia exposure. Correlations among sirtuins' expression and clinical and molecular parameters were also analyzed. We found that sirtuin1-6 exhibited significant higher mRNA expression in visceral adipose-derived stem cells compared to subcutaneous adipose-derived stem cells of non-obese subjects. Sirtuin1-6 levels were markedly reduced in visceral adipose-derived stem cells of obese patients. Sirtuins' expression in visceral adipose-derived stem cells correlated negatively with body mass index and C-reactive protein and positively with peroxisome proliferator-activated receptor delta. Finally, only in the visceral adipose-derived stem cells of obese patients hypoxia-induced mRNA expression of all of the sirtuins. Our results highlight that sirtuins' levels in adipose-derived stem cells are consistent with protective effects against visceral obesity and inflammation, and suggest a transcriptional mechanism through which acute hypoxia up-regulates sirtuins in the visceral adipose-derived stem cells of obese patients

Cooperative Interactions between PBX, PREP, and HOX Proteins Modulate the Activity of the α2(V) Collagen (COL5A2) Promoter

Cell type-specific expression of the human alpha2(V) collagen (COL5A2) gene depends on a cis-acting element that consists of two contiguous protein binding sites (FPA and FPB) located between nucleotides -149 and -95, relative to the transcription start site. The present study focused on the characterization of the FPB-bound complex. DNA binding assays and cell transfection experiments revealed that the bipartite core sequence of FPB (5'-ATCAATCA-3') binds the PBX1/2, PREP1, and HOXB1 proteins, and this in turn leads to promoter transactivation. In the presence of all three nuclear factors, cooperative interactions between recombinant PBX1 and PREP1 or PBX1 and HOXB1 result in binding of the heterodimers to FPB in vitro. Similarly, overexpression of different combinations of PBX1, PREP1, and HOXB1 transactivates FPB-driven transcription. In contrast to the composition of the FPB complex purified from COL5A2-positive cells, the FPB complex from COL5A2-negative cells contains PBX2 and PREP1 but lacks PBX1. However, PBX1 exogenously introduced into COL5A2-negative cells cannot stimulate FPB-driven transcription unless co-expressed with PREP1. Within the intrinsic limitations of the experimental model, our results indicate that combinatorial interactions among PBX and PREP or HOX proteins are involved in regulating tissue-specific production of collagen V

Enhanced Healing of Diabetic Wounds by Topical Administration of Adipose Tissue-Derived Stromal Cells Overexpressing Stromal-Derived Factor-1: Biodistribution and Engraftment Analysis by Bioluminescent Imaging

Chronic ulcers represent a major health problem in diabetic patients resulting in pain and discomfort. Conventional therapy does not guarantee adequate wound repair. In diabetes, impaired healing is partly due to poor endothelial progenitor cells mobilisation and homing, with altered levels of the chemokine stromal-derived factor-1 (SDF-1) at the wound site. Adipose tissue-associated stromal cells (AT-SCs) can provide an accessible source of progenitor cells secreting proangiogenic factors and differentiating into endothelial-like cells. We demonstrated that topical administration of AT-SCs genetically modified ex vivo to overexpress SDF-1, promotes wound healing into diabetic mice. In particular, by in vivo bioluminescent imaging analysis, we monitored biodistribution and survival after transplantation of luciferase-expressing cells. In conclusion, this study indicates the therapeutic potential of AT-SCs administration in wound healing, through cell differentiation, enhanced cellular recruitment at the wound site, and paracrine effects associated with local growth-factors production

Caregiver burden and its related factors in advanced Parkinson’s disease: data from the PREDICT study

Introduction: Caring for a person with Parkinson’s disease (PD) is associated with an increased risk of psychiatric morbidity and persistent distress. The objective of this study was to describe the burden and the related factors of caregivers of advanced PD (APD) patients either treated with continuous dopaminergic delivery systems or standard therapy. Methods: This cross-sectional, epidemiologic study conducted in 13 Italian sites enrolled PD patients treated with continuous dopaminergic delivering systems [either levodopa/carbidopa intestinal gel (LCIG) infusion or continuous subcutaneous apomorphine infusion (CSAI)] or continuation of standard of care (SOC) with a caregiver. Patient quality of life (QoL) and caregiver burden were assessed using the Parkinson’s Disease Questionnaire (PDQ-8) and Zarit Burden Inventory (ZBI), respectively. Results: 126 patients (mean age 69.3 ± 8 years) and their caregivers (mean age 57.9 ± 12.9) were enrolled. Most caregivers were spouses. Fifty-three patients were treated with LCIG, 19 with CSAI, and 54 with SOC. Mean ZBI scores were 29.6 ± 14.4 for LCIG, 35.8 ± 20.2 for CSAI, and 31.4 ± 16.0 for SOC. Caregivers of LCIG, CSAI, and SOC patients showed no burden or mild/moderate burden in 74, 53, and 63% of the cases, respectively. Mean PDQ-8 scores were 11.25 ± 5.67, 11.26 ± 5.55, and 14.22 ± 6.51 in LCIG, CSAI, and SOC patients. Neurologists considered patients “very much or much improved” in 89, 58, and 13% of the LCIG, CSAI, and SOC groups using the Clinical Global Impression–Global Improvement Scale. Predictors significantly associated with caregiver burden were patients and caregivers’ judgment of QoL and caregivers’ need to change work. Conclusions: Caregiver burden showed a tendency to be lower when patients are treated with LCIG than with CSAI or SOC

Towards Therapeutic Delivery of Extracellular Vesicles: Strategies for In Vivo Tracking and Biodistribution Analysis

Extracellular vesicles (EVs), such as microvesicles and exosomes, are membranous structures containing bioactive material released by several cells types, including mesenchymal stem/stromal cells (MSCs). Increasing lines of evidences point to EVs as paracrine mediators of the beneficial effects on tissue remodeling associated with cell therapy. Administration of MSCs-derived EVs has therefore the potential to open new and safer therapeutic avenues, alternative to cell-based approaches, for degenerative diseases. However, an enhanced knowledge about in vivo EVs trafficking upon delivery is required before effective clinical translation. Only a few studies have focused on the biodistribution analysis of exogenously administered MSCs-derived EVs. Nevertheless, current strategies for in vivo tracking in animal models have provided valuable insights on the biodistribution upon systemic delivery of EVs isolated from several cellular sources, indicating in liver, spleen, and lungs the preferential target organs. Different strategies for targeting EVs to specific tissues to enhance their therapeutic efficacy and reduce possible off-target effects have been investigated. Here, in the context of a possible clinical application of MSC-derived EVs for tissue regeneration, we review the existing strategies for in vivo tracking and targeting of EVs isolated from different cellular sources and the studies elucidating the biodistribution of exogenously administered EVs

Protein disulfide isomerase as a prosurvival factor in cell therapy for muscular and vascular diseases

Abstract Background Cell therapy for degenerative diseases aims at rescuing tissue damage by delivery of precursor cells. Thus far, this strategy has been mostly unsuccessful due to massive loss of donor cells shortly after transplantation. Several strategies have been applied to increase transplanted cell survival but only with limited success. The endoplasmic reticulum (ER) is an organelle involved in protein folding, calcium homeostasis, and lipid biosynthesis. Protein disulfide isomerase (PDI) is a molecular chaperone induced and activated by ER stress. PDI is induced by hypoxia in neuronal, cardiac, and endothelial cells, supporting increased cell survival to hypoxic stress and protection from apoptosis in response to ischemia. Methods We achieved ex vivo PDI gene transfer into luciferase-expressing myoblasts and endothelial cells. We assessed cell engraftment upon intramuscular transplantation into a mouse model of Duchenne muscular dystrophy (mdx mouse) and into a mouse model of ischemic disease. Results We observed that loss of full-length dystrophin expression in mdx mice muscle leads to an increase of PDI expression, possibly in response to augmented ER protein folding load. Moreover, we determined that overexpression of PDI confers a survival advantage for muscle cells in vitro and in vivo to human myoblasts injected into murine dystrophic muscle and to endothelial cells administered upon hindlimb ischemia damage, improving the therapeutic outcome of the cell therapy treatment. Conclusions Collectively, these results suggest that overexpression of PDI may protect transplanted cells from hypoxia and other possibly occurring ER stresses, and consequently enhance their regenerative properties

Challenges and strategies for improving the regenerative effects of mesenchymal stromal cell-based therapies

Cell-based therapies have the potential to revolutionize current treatments for diseases with high prevalence and related economic and social burden. Unfortunately, clinical trials have made only modest improvements in restoring normal function to degenerating tissues. This limitation is due, at least in part, to the death of transplanted cells within a few hours after transplant due to a combination of mechanical, cellular, and host factors. In particular, mechanical stress during implantation, extracellular matrix loss upon delivery, nutrient and oxygen deprivation at the recipient site, and host inflammatory response are detrimental factors limiting long-term transplanted cell survival. The beneficial effect of cell therapy for regenerative medicine ultimately depends on the number of administered cells reaching the target tissue, their viability, and their promotion of tissue regeneration. Therefore, strategies aiming at improving viable cell engraftment are crucial for regenerative medicine. Here we review the major factors that hamper successful cell engraftment and the strategies that have been studied to enhance the beneficial effects of cell therapy. Moreover, we provide a perspective on whether mesenchymal stromal cell-derived extracellular vesicle delivery, as a cell-free regenerative approach, may circumvent current cell therapy limitations