Hyaluronan expression following middle cerebral artery occlusion in the rat

Hyaluronic acid, a major component of the brain extracellular matrix, is a regulator of angiogenesis, cell differentiation and migration. We used the rat middle cerebral artery occlusion model to show hyaluronan accumulation in stroke-affected areas. Using reverse transcription-polymerase chain reaction and Western blotting we showed up-regulation of hyaluronidase-1 and 2 between 1 h and 21 days after stroke. Hyaluronidase-1 was up-regulated earlier than hyaluronidase-2. The hyaladherins, receptor for hyaluronan-mediated motility and CD44 were also increased after stroke. Using immunohistochemistry, we showed association of hyaluronidases 1/2 and hyaladherins with neurons in the infarcted and peri-infarcted regions and hyaluronidase-1 with microvessels. Hyaluronan synthesis and degradation in the stroke hemisphere might have an impact on neuronal survival, angiogenesis and general tissue remodelling after stroke

Changes in hyaluronan production and metabolism following ischaemic stroke in man

The extent of recovery from stroke is dependent on the survival of neurons, particularly in peri-infarcted regions. Angiogenesis is critical for the development of new microvessels and leads to re-formation of collateral circulation, reperfusion and better recovery. Hyaluronan (HA) is an important component of the brain extracellular matrix and a regulator of cellular differentiation, migration, proliferation and angiogenesis. We have found that the production of total HA and low molecular mass 3-10 disaccharides of HA (o-HA) was increased in post-mortem tissue and in the serum of patients 1, 3, 7 and 14 days (peaking at 7 days) after ischaemic stroke. Hyaluronidase activity was also increased in serum samples (peaking after 3 days), which might explain the subsequent increase in o-HA. Affinity-histochemical staining was performed using a HA-specific biotinylated binding protein, and it showed enhanced deposition of HA in blood vessels and intracellularly as well as in the nuclei of peri-infarcted neurons. Western blotting and immunohistochemistry demonstrated upregulation of HA synthases (HAS1 and 2) and hyaluronidases (HYAL1 and 2) in inflammatory cells from both stroke and peri-infarcted regions of the brain. HYAL1 was upregulated in microvesssels and intracellularly in neurons, whilst HAS2 became translocated into the nuclei of neurons in peri-infarcted areas. Receptor for HA-mediated motility was observed intracellularly and in the nuclei of neurons, in the tunica media of larger blood vessels and in the endothelial cells of microvessels in stroke-affected tissue, whilst expression of other receptors for HA, CD44 and tumour necrosis factor-stimulated gene 6 (TSG-6) were mainly increased in infiltrating mononuclear cells from inflammatory regions. The data presented here demonstrate that HA breakdown is a feature of the acute stage of stroke injury. Increased o-HA production soon after stroke may be detrimental through enhancement of the inflammatory response, whilst activation of HA and/or o-HA-induced cellular signalling pathways in neurons and microvessels may impact on the remodelling process by stimulating angiogenesis and revascularization, as well as the survival of susceptible neurons

Airway Smooth Muscle Cells Synthesize Hyaluronan Cable Structures Independent of Inter-α-inhibitor Heavy Chain Attachment*S⃞

The covalent association of inter-α-inhibitor-derived heavy chains (HCs) with hyaluronan was first described in synovial fluid from arthritic patients and later described as a structural and functional component of hyaluronan “cable” structures produced by many different cells and stimuli. HC transfer has been shown to be mediated by the protein product of TSG-6 (tumor necrosis factor-stimulated gene 6). Considering the accumulation of hyaluronan in airways following asthmatic attacks and the subsequent infiltration of leukocytes, we sought to characterize HC substitution of hyaluronan “cables” in primary mouse airway smooth muscle cells (MASM) and primary human airway smooth muscle cells (HASM). We found that cells derived from mice lacking TSG-6 had no defect in hyaluronan production or hyaluronan-mediated leukocyte adhesion when treated with the viral mimic poly(I,C). Functional hyaluronan cables were induced by cycloheximide in the confirmed absence of protein synthesis, with or without simultaneous treatment with poly(I,C). We characterized the species specificity of the antibody other investigators used to describe the HC-hyaluronan complex of hyaluronan cables and found minimal affinity to bovine-derived HCs in contrast to HCs from mouse and human sera. Thus, we cultured MASM and HASM cells in serum from these three sources and analyzed hyaluronan extracts for HCs and other hyaluronan-binding proteins, using parallel cumulus cell-oocyte complex (COC) extracts as positive controls. We conclude that, if hyaluronan cables derived from MASM and HASM cells are substituted with HCs, the amount of substitution is significantly below the limit of detection when compared with COC extracts of similar hyaluronan mass

Stress-dependent Daxx-CHIP Interaction Suppresses the p53 Apoptotic Program*♦

Our previous studies have implicated CHIP (carboxyl terminus of Hsp70-interacting protein) as a co-chaperone/ubiquitin ligase whose activities yield protection against stress-induced apoptotic events. In this report, we demonstrate a stress-dependent interaction between CHIP and Daxx (death domain-associated protein). This interaction interferes with the stress-dependent association of HIPK2 with Daxx, blocking phosphorylation of serine 46 in p53 and inhibiting the p53-dependent apoptotic program. Microarray analysis confirmed suppression of the p53-dependent transcriptional portrait in CHIP+/+ but not in CHIP−/− heat shocked mouse embryonic fibroblasts. The interaction between CHIP and Daxx results in ubiquitination of Daxx, which is then partitioned to an insoluble compartment of the cell. In vitro ubiquitination of Daxx by CHIP revealed that ubiquitin chain formation utilizes non-canonical lysine linkages associated with resistance to proteasomal degradation. The ubiquitination of Daxx by CHIP utilizes lysines 630 and 631 and competes with the sumoylation machinery of the cell at these residues. These studies implicate CHIP as a stress-dependent regulator of Daxx that counters the pro-apoptotic influence of Daxx in the cell. By abrogating p53-dependent apoptotic pathways and by ubiquitination competitive with Daxx sumoylation, CHIP integrates the proteotoxic stress response of the cell with cell cycle pathways that influence cell survival

Human Recombinant Hyaluronidase Injections For Upper Limb Muscle Stiffness in Individuals With Cerebral Injury: A Case Series

Spasticity, muscle stiffness and contracture cause severe disability after central nervous system injury. However, current treatment options for spasticity produce muscle weakness which can impede movement, and do not directly address muscle stiffness. Here we propose that the accumulation of hyaluronan within muscles promotes the development of muscle stiffness, and report that treatment with the enzyme hyaluronidase increases upper limb movement and reduces muscle stiffness without producing weakness. 20 patients with unilateral upper limb spasticity received multiple intramuscular injections of human recombinant hyaluronidase with saline at a single visit. The safety and efficacy of the injections, passive and active movement, and muscle stiffness at eight upper limb joints were assessed at four time points: pre-injection (T0), within 2 weeks (T1), within 4–6 weeks (T2), and within 3–5 months post-injection (T3). There were no clinically significant adverse effects from the injections. Passive movement at all joints, and active movement at most joints increased at T1, and persisted at T2 and T3 for most joints. The modified Ashworth scores also declined significantly over time post-injection. Hyaluronidase injections offer a safe and potentially efficacious treatment for muscle stiffness in neurologically impaired individuals. These results warrant confirmation in placebo-controlled clinical trials

Poly(methacrylated hyaluronan-co-ethyl acrylate) copolymer networks with tunable properties and enzymatic degradation

[EN] Semi-biodegradable copolymer networks derived from two immiscible components, a hyaluronan derivative (methacrylated hyaluronan) and poly(ethyl acrylate), have been developed for soft tissue engineering applications. These copolymer networks combine the hydrogel-like properties of hyaluronan networks with the rubber-like behavior and low water uptake of the poly(ethyl acrylate) networks. In order to copolymerize hyaluronan and ethyl acrylate, a hyaluronan derivative containing vinyl groups was first obtained. In spite of the different nature of both polymers, the variation of the weight fraction of ethyl acrylate in the copolymerization yields copolymer networks with co-continuity of phases, and tunable swelling degree and mechanical properties. In addition, the MeHA chains are susceptible of being enzymatically degraded, and the resulting materials are mainly composed of a PEA framework with structural integrity. These copolymers networks are good candidates to assist the regeneration process of soft tissues when longer residence times are required.Support of this work through projects PRI-PIMNEU-2011 -1372 (ERANET-Neuron) and MAT2011-28791-C03-02 is gratefully acknowledged.Perez Garnes, M.; Monleón Pradas, M. (2017). Poly(methacrylated hyaluronan-co-ethyl acrylate) copolymer networks with tunable properties and enzymatic degradation. Polymer Degradation and Stability. 144:241-250. https://doi.org/10.1016/j.polymdegradstab.2017.08.025S24125014