Donor Sites and Harvesting Techniques Affect miRNA Cargos of Extracellular Vesicles Released by Human Adipose-Derived Mesenchymal Stromal Cells

Osteoarthritis (OA) is a degenerative joint disorder characterized by the progressive deterioration of articular cartilage driven and sustained by catabolic and inflammatory processes that lead to pain and functional impairment. Adipose-derived stem cells (ASCs) have emerged as a promising therapeutic strategy for OA due to their regenerative potential, which mainly relies on the adaptive release of paracrine molecules that are soluble or encapsulated in extracellular vesicles (EVs). The biological effects of EVs specifically depend on their cargo; in particular, microRNAs (miRNAs) can specifically modulate target cell function through gene expression regulation. This study aimed to investigate the impact of collection site (abdominal vs. peri-trochanteric adipose tissue) and collection method (surgical excision vs. lipoaspiration) on the miRNAs profile in ASC-derived EVs and their potential implications for OA therapy. EV-miRNA cargo profiles from ASCs of different origins were compared. An extensive bioinformatics search through experimentally validated and OA-related targets, pathways, and tissues was conducted. Several miRNAs involved in the restoration of cartilage homeostasis and in immunomodulation were identified in all ASC types. However, EV-miRNA expression profiles were affected by both the tissue-harvesting site and procedure, leading to peculiar characteristics for each type. Our results suggest that adipose-tissue-harvesting techniques and the anatomical site of origin influence the therapeutic efficacy of ASC-EVs for tissue-specific regenerative therapies in OA, which warrants further investigation

Stathmins and Motor Neuron Diseases: Pathophysiology and Therapeutic Targets

Motor neuron diseases (MNDs) are a group of fatal, neurodegenerative disorders with different etiology, clinical course and presentation, caused by the loss of upper and lower motor neurons (MNs). MNs are highly specialized cells equipped with long, axonal processes; axonal defects are some of the main players underlying the pathogenesis of these disorders. Microtubules are key components of the neuronal cytoskeleton characterized by dynamic instability, switching between rapid polymerization and shrinkage. Proteins of the stathmin family affect microtubule dynamics regulating the assembly and the dismantling of tubulin. Stathmin-2 (STMN2) is one of the most abundantly expressed genes in MNs. Following axonal injury, STMN2 expression is upregulated, and the protein is transported toward the growth cones of regenerating axons. STMN2 has a critical role in axonal maintenance, and its dysregulation plays an important role in neurodegenerative processes. Stathmin-1 (STMN1) is a ubiquitous protein that is highly expressed during the development of the nervous system, and its phosphorylation controls microtubule dynamics. In the present review, we summarize what is currently known about the involvement of stathmin alterations in MNDs and the potential therapeutic effect of their modulation, with a specific focus on the most common forms of MND, amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA)

Hyperglycaemia significantly and in a dose-dependent fashion affected neurite outgrowth.

<p>(A) Exposure to increasing glucose concentrations induced a significant dose-dependent decrease of neurite outgrowth in neuron-SC co-cultures (B) but not in neuron monocultures. (C) Exposure of neuron monocultures to hyperglycaemia-conditioned SC monoculture medium caused a significant decrease of the neurite outgrowth. All data are normalized to control and presented as mean±SEM of independent experiments (<i>n</i> = 8) *p<0.05; **p<0.005; ***p<0.0005 vs control. (D) Cytokine array of hyperglycaemia-conditioned SC monoculture medium showing increase of VEGF compared to control. (E) Data are expressed as relative levels of VEGF.</p

Bevacizumab prevented and restored peripheral nerve functions in diabetic rats.

<p>Control and STZ-diabetic rats were treated with bevacizumab according to the prevention (A, B, C) or therapeutic (D, E, F) schedule. (A) Bevacizumab prevented in a dose-dependent fashion thermal hypoalgesia, (B) mechanical threshold decrease and (C) nerve conduction velocity decrease in diabetic rats. In the therapeutic schedule, bevacizumab restored (D) thermal hypoalgesia (E) mechanical threshold and (F) nerve conduction velocity decrease in diabetic rats. Data are expressed as mean±SEM (<i>n</i> = 8 animals per group) *p<0.05 vs controls; **p<0.01 vs controls; ****p<0.001 vs controls; #*p<0.05 vs STZ; ##p<0.01 vs STZ.</p

Hyperglycaemia did not increase DRG neuron or Schwann cell (SC) apoptosis.

<p>(A) Neuron-SC co-cultures showed a modest increase of apoptosis rate only at the highest glucose concentration; paclitaxel (tax) and cisplatin (cis) exposition was used as positive controls. (B) In DRG neuron monocultures, hyperglycaemia did not induce apoptosis. (C) Tubulin-III and GFAP staining demonstrated that apoptosis mainly involved SC. (D) SC monocultures showed a mild increase of apoptosis rate at the highest glucose concentrations, similar to that observed in co-cultures (see C). (E) Flow cytometry by annexin V/PI assay confirmed the absence of apoptosis in co-cultures exposed to hyperglycaemia. (F) Representative cytogram showing the absence of apoptosis in control co-culture (ctrl) and after 24 hour exposition to hyperglycaemia 45 mM (hg), compared to the high apoptosis rate after exposition to anti-neoplastic compounds (tax, cis). Data are expressed as mean±SEM of independent experiments (<i>n</i> = 8) *p<0.05; **p<0.005; ***p<0.0005 vs controls; #p<0.05; ##p<0.005; ###p<0.0005 vs SC controls.</p

Hyperglycaemia-induced post-translational regulation of VEGF.

<p>(A) Exposure to glucose 45 mM for 24 hours increased the level of secreted VEGF in neuron-SC co-cultures and SC monocultures. Data are express as percentage difference between their own control in independent experiments (<i>n</i> = 5) *p<0.05. (B) Hyperglycaemia significantly increased VEGF mRNA only in neuron monocultures (<i>n</i> = 5). (C) Representative and (D) quantitative western blot (WB) demonstrating VEGF decrease in hyperglycaemia-conditioned SC monocultures compared with control SC monocultures. VEGF level was not affected in neuron monocultures. Data are expressed as mean±SEM of independent experiments (<i>n</i> = 4) *p<0.05 vs controls. (E) VEGF induced a significant reduction in axonal outgrowth in neuron/Schwann cells coculture. Data are expressed as mean±SEM of independent experiments (<i>n</i> = 3) *p<0.05 vs controls.</p

Bevacizumab prevented hyperglycaemia-mediated neurite outgrowth impairment and normalized FLT-1.

<p>(A) bevacizumab prevented the decrease of hyperglycaemia-mediated neurite outgrowth in neuron-SC co-cultures (<i>n</i> = 5) and (B) the increase of FLT-1 mRNA in neurons monocultures (<i>n</i> = 5). (C) Representative and (D) quantitative western blot (WB) (<i>n</i> = 5) showing the significant decrease of FLT-1 in hyperglycaemia-conditioned SC monocultures and the preventive effect of bevacizumab. (E) sFLT-1 decreased in the medium of all neuron and SC monocultures and co-cultures exposed to hyperglycaemia (<i>n</i> = 5). (F) Bevacizumab did not affect sFLT-1 level in hyperglycaemia-conditioned co-cultures (<i>n</i> = 3). Data are expressed as mean±SEM of independent experiments. *p<0.05 vs controls; #p<0.05 vs hyperglycaemia; ##p<0.01 vs hyperglycaemia.</p

Jagged Ligands Enhance the Pro-Angiogenic Activity of Multiple Myeloma Cells

Multiple myeloma (MM) is an incurable plasma cell malignancy arising primarily within the bone marrow (BM). During MM progression, different modifications occur in the tumor cells and BM microenvironment, including the angiogenic shift characterized by the increased capability of endothelial cells to organize a network, migrate and express angiogenic factors, including vascular endothelial growth factor (VEGF). Here, we studied the functional outcome of the dysregulation of Notch ligands, Jagged1 and Jagged2, occurring during disease progression, on the angiogenic potential of MM cells and BM stromal cells (BMSCs). Jagged1&ndash;2 expression was modulated by RNA interference or soluble peptide administration, and the effects on the MM cell lines&rsquo; ability to induce human pulmonary artery cells (HPAECs) angiogenesis or to indirectly increase the BMSC angiogenic potential was analyzed in vitro; in vivo validation was performed on a zebrafish model and MM patients&rsquo; BM biopsies. Overall, our results indicate that the MM-derived Jagged ligands (1) increase the tumor cell angiogenic potential by directly triggering Notch activation in the HPAECs or stimulating the release of angiogenic factors, i.e., VEGF; and (2) stimulate the BMSCs to promote angiogenesis through VEGF secretion. The observed pro-angiogenic effect of Notch activation in the BM during MM progression provides further evidence of the potential of a therapy targeting the Jagged ligands

Combinatorial strategies targeting NEAT1 and AURKA as new potential therapeutic options for multiple myeloma

Multiple myeloma (MM) is a dreadful disease, marked by the uncontrolled proliferation of clonal plasma cells (PCs) within the bone marrow (BM). MM is characterized by a highly heterogeneous clinical and molecular background, supported by severe genomic alterations. Important deregulation of long non-coding RNAs (lncRNAs) expression has been reported in MM patients, influencing progression and therapy resistance. NEAT1 is a lncRNA essential for nuclear paraspeckles and involved in gene expression regulation. We showed that NEAT1 supports MM proliferation making this lncRNA an attractive therapeutic candidate. Here, we used a combinatorial strategy integrating transcriptomic and computational approaches with functional high-throughput drug screening, to identify compounds that synergize with NEAT1 inhibition in restraining MM cells growth. AUKA inhibitors were identified as top-scoring drugs in these analyses. We showed that the combination of NEAT1 silencing and AURKA inhibitors in MM profoundly impairs microtubule organization and mitotic spindle assembly, finally leading to cell death. Analysis of the large publicly CoMMpass dataset showed that in MM patients AURKA expression is strongly associated with reduced progression-free (p &lt; 0.0001) and overall survival probability (p &lt; 0.0001) and patients displaying high expression levels of both NEAT1 and AURKA have a worse clinical outcome. Finally, using RNA-sequencing data from NEAT1 knockdown (KD) MM cells, we identified the AURKA allosteric regulator TPX2 as a new NEAT1 target in MM and as a mediator of the interplay between AURKA and NEAT1, therefore providing a possible explanation of the synergistic activity observed upon their combinatorial inhibition