title: Pharmacological inhibition of caspase and calpain proteases: a novel strategy to enhance the homing responses of cord blood HSPCs during expansion.
abstract: BACKGROUND
  Expansion of hematopoietic stem/progenitor cells (HSPCs) is a well-known strategy employed to facilitate the transplantation outcome. We have previously shown that the prevention of apoptosis by the inhibition of cysteine proteases, caspase and calpain played an important role in the expansion and engraftment of cord blood (CB) derived HSPCs. We hypothesize that these protease inhibitors might have maneuvered the adhesive and migratory properties of the cells rendering them to be retained in the bone marrow for sustained engraftment. The current study was aimed to investigate the mechanism of the homing responses of CB cells during expansion.
  
  
  RESULTS
  CB derived CD34(+) cells were expanded using a combination of growth factors with and without Caspase inhibitor -zVADfmk or Calpain 1 inhibitor- zLLYfmk. The cells were analyzed for the expression of homing-related molecules. In vitro adhesive/migratory interactions and actin polymerization dynamics of HSPCs were assessed. In vivo homing assays were carried out in NOD/SCID mice to corroborate these observations. We observed that the presence of zVADfmk or zLLYfmk (inhibitors) caused the functional up regulation of CXCR4, integrins, and adhesion molecules, reflecting in a higher migration and adhesive interactions in vitro. The enhanced actin polymerization and the RhoGTPase protein expression complemented these observations. Furthermore, in vivo experiments showed a significantly enhanced homing to the bone marrow of NOD/SCID mice.
  
  
  CONCLUSIONS
  Our present study reveals another novel aspect of the regulation of caspase and calpain proteases in the biology of HSPCs. The priming of the homing responses of the inhibitor-cultured HSPCs compared to the cytokine-graft suggests that the modulation of these proteases may help in overcoming the major homing defects prevalent in the expansion cultures thereby facilitating the manipulation of cells for transplant procedures.
authors.affiliation: Stem Cell Biology Laboratory, National Centre for Cell Science, Ganeshkhind, Pune University Campus, Pune, Maharashtra, India.
authors.forename: V M
authors.initials: VM
authors.lastname: Sangeetha
authors.forename: Darshana
authors.initials: D
authors.lastname: Kadekar
authors.forename: Vaijayanti P
authors.initials: VP
authors.lastname: Kale
authors.forename: Lalita S
authors.initials: LS
authors.lastname: Limaye
mesh_terms: D000199:Actins / Q000737:chemistry; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D002154:Calpain / Q000037:antagonists & inhibitors*; D061945:Caspase Inhibitors; D002448:Cell Adhesion / Q000187:drug effects; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects; D016764:Cell Polarity / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D054377:Chemokine CXCL12 / Q000235:genetics / Q000378:metabolism; D005260:Female; D005312:Fetal Blood / Q000166:cytology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D039441:Integrin alpha4 / Q000378:metabolism; D039482:Integrin alpha5 / Q000378:metabolism; D051379:Mice; D011480:Protease Inhibitors / Q000494:pharmacology*; D055503:Protein Multimerization / Q000187:drug effects; D020836:Protein Structure, Quaternary; D019718:Receptors, CXCR4 / Q000235:genetics / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism
chemical_list: D000199:Actins; D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D015815:Cell Adhesion Molecules; D054377:Chemokine CXCL12; D039482:Integrin alpha5; D011480:Protease Inhibitors; D019718:Receptors, CXCR4; C484771:benzyloxycarbonyl-valyl-aspartic acid fluoromethyl ketone; D039441:Integrin alpha4; D002154:Calpain; D020741:rho GTP-Binding Proteins