Anti-HAPLN1 monoclonal antibody, clone 427220

In the rodent model of temporal lobe epilepsy, there is extensive synaptic reorganization within the hippocampus following a single prolonged seizure event, after which animals eventually develop epilepsy. The perineuronal net (PN), a component of the neural extracellular matrix (ECM), primarily surrounds inhibitory interneurons and, under normal conditions, restricts synaptic reorganization. The objective of the current study was to explore the effects of status epilepticus (SE) on PNs in the adult hippocampus. The aggrecan component of the PN was studied, acutely (48 h post-SE), sub-acutely (1 week post-SE) and during the chronic period (2 months post-SE). Aggrecan expressing PNs decreased by 1 week, likely contributing to a permissive environment for neuronal reorganization, and remained attenuated at 2 months. The SE-exposed hippocampus showed many PNs with poor structural integrity, a condition rarely seen in controls. Additionally, the decrease in the aggrecan component of the PN was preceded by a decrease in hyaluronan and proteoglycan link protein 1 (HAPLN1) and hyaluronan synthase 3 (HAS3), which are components of the PN known to stabilize the connection between aggrecan and hyaluronan, a major constituent of the ECM. These results were replicated in vitro with the addition of excess KCl to hippocampal cultures. Enhanced neuronal activity caused a decrease in aggrecan, HAPLN1 and HAS3 around hippocampal cells in vivo and in vitro, leaving inhibitory interneurons susceptible to increased synaptic reorganization. These studies are the foundation for future experiments to explore how loss of the PN following SE contributes to the development of epilepsy.

Objective: To determine the optimal environmental conditions for chondrogenic differentiation of human adipose tissue-derived mesenchymal stromal/stem cells (AMSCs). In this investigation we specifically investigate the role of oxygen tension and 3-dimensional (3D) culture systems. Design: Both AMSCs and primary human chondrocytes were cultured for 21 days in chondrogenic media under normoxic (21% oxygen) or hypoxic (2% oxygen) conditions using 2 distinct 3D culture methods (high-density pellets and poly-epsilon-caprolactone [PCL] scaffolds). Histologic analysis of chondro-pellets and the expression of chondrocyte-related genes as measured by reverse transcriptase quantitative polymerase chain reaction were used to evaluate the efficiency of differentiation. Results: AMSCs are capable of expressing established cartilage markers including COL2A1, ACAN, and DCN when grown in chondrogenic differentiation media as determined by gene expression and histologic analysis of cartilage markers. Expression of several cartilage-related genes was enhanced by low oxygen tension, including ACAN and HAPLN1. The pellet culture environment also promoted the expression of hypoxia-inducible cartilage markers compared with cells grown on 3D scaffolds. Conclusions: Cell type-specific effects of low oxygen and 3D environments indicate that mesenchymal cell fate and differentiation potential is remarkably sensitive to oxygen. Genetic programming of AMSCs to a chondrocytic phenotype is effective under hypoxic conditions as evidenced by increased expression of cartilage-related biomarkers and biosynthesis of a glycosaminoglycan-positive matrix. Lower local oxygen levels within cartilage pellets may be a significant driver of chondrogenic differentiation.