Mesenchymal stem cells have been used for cardiovascular regenerative therapy for decades. These cells have been established as one of the potential therapeutic agents, following several tests in animal models and clinical trials. In the process, various sources of mesenchymal stem cells have been identified which help in cardiac regeneration by either revitalizing the cardiac stem cells or revascularizing the arteries and veins of the heart. Although mesenchymal cell therapy has achieved considerable admiration, some challenges still remain that need to be overcome in order to establish it as a successful technique. This in-depth review is an attempt to summarize the major sources of mesenchymal stem cells involved in myocardial regeneration, the significant mechanisms involved in the process with a focus on studies (human and animal) conducted in the last 6 years and the challenges that remain to be addressed.

We hypothesized that the AAV2 vector is targeted for destruction in the cytoplasm by the host cellular kinase/ubiquitination/proteasomal machinery and that modification of their targets on AAV2 capsid may improve its transduction efficiency. In vitro analysis with pharmacological inhibitors of cellular serine/threonine kinases (protein kinase A, protein kinase C, casein kinase II) showed an increase (20–90%) on AAV2-mediated gene expression. The three-dimensional structure of AAV2 capsid was then analyzed to predict the sites of ubiquitination and phosphorylation. Three phosphodegrons, which are the phosphorylation sites recognized as degradation signals by ubiquitin ligases, were identified. Mutation targets comprising eight serine (S) or seven threonine (T) or nine lysine (K) residues were selected in and around phosphodegrons on the basis of their solvent accessibility, overlap with the receptor binding regions, overlap with interaction interfaces of capsid proteins, and their evolutionary conservation across AAV serotypes. AAV2-EGFP vectors with the wild-type (WT) capsid or mutant capsids (15 S/T→alanine [A] or 9 K→arginine [R] single mutant or 2 double K→R mutants) were then evaluated in vitro. The transduction efficiencies of 11 S/T→A and 7 K→R vectors were significantly higher (∼63–90%) than the AAV2-WT vectors (∼30–40%). Further, hepatic gene transfer of these mutant vectors in vivo resulted in higher vector copy numbers (up to 4.9-fold) and transgene expression (up to 14-fold) than observed from the AAV2-WT vector. One of the mutant vectors, S489A, generated ∼8-fold fewer antibodies that could be cross-neutralized by AAV2-WT. This study thus demonstrates the feasibility of the use of these novel AAV2 capsid mutant vectors in hepatic gene therapy.

Background The present study was designed to investigate the molecular signaling events from onset of bleeding through the development of arthropathy in a murine model of hemophilia A. Methods and results A sharp‐injury model of hemarthrosis was used. A global gene expression array on joint‐specific RNA isolated 3 h post‐injury revealed nuclear factor‐kappa B (NF‐κB) as the major transcription factor triggering inflammation. As a number of genes encoding the cytokines, growth factors and hypoxia regulating factors are known to be activated by NF‐κB and many of these are part of the pathogenesis of various joint diseases, we reasoned that NF‐κB‐associated pathways may play a crucial role in blood‐induced joint damage. To further understand its role, we screened NF‐κB‐associated pathways between 1 h to 90 days after injury. After a single articular bleed, distinct members of the NF‐κB family (NF‐κB1/NF‐κB2/RelA/RelB) and their responsive pro‐inflammatory cytokines (IL−1β/IL−6/IFNγ/TNFα) were significantly up‐regulated (> 2 fold, P < 0.05) in injured vs. control joints at the various time‐points analyzed (1 h/3 h/7 h/24 h). After multiple bleeds (days 30/60/75/90), there was increased expression of NF‐κB‐associated factors that contribute to hypoxia (HIF‐1α, 3.3−6.5 fold), angiogenesis (VEGF‐α, 2.5−4.4 fold) and chondrocyte damage (matrix metalloproteinase−13, 2.8−3.8 fold) in the injured joints. Micro RNAs (miR) that are known to regulate NF‐κB activation (miRs‐9 and 155), inflammation (miRs‐16, 155 and 182) and apoptosis (miRs‐19a, 155 and 186) were also differentially expressed (−4 to +13‐fold) after joint bleeding, indicating that the small RNAs could modulate the arthropathy phenotype. Conclusions These data suggest that NF‐κB‐associated signaling pathways are involved in the development of hemophilic arthropathy.