Sufan Chien

MicroRNA-155 potentiates the inflammatory response in hypothermia by suppressing IL-10 production

Therapeutic hypothermia is commonly used to improve neurological outcomes in patients after cardiac arrest. However, therapeutic hypothermia increases sepsis risk and unintentional hypothermia in surgical patients increases infectious complications. Nonetheless, the molecular mechanisms by which hypothermia dysregulates innate immunity are incompletely understood. We found that exposure of human monocytes to cold (32°C) potentiated LPS‐induced production of TNF and IL‐6, while blunting IL‐10 production. This dysregulation was associated with increased expression of microRNA‐155 (miR‐155), which potentiates Toll‐like receptor (TLR) signaling by negatively regulating Ship1 and Socs1. Indeed, Ship1 and Socs1 were suppressed at 32°C and miR‐155 antagomirs increased Ship1 and Socs1 and reversed the alterations in cytokine production in cold‐exposed monocytes. In contrast, miR‐155 mimics phenocopied the effects of cold exposure, reducing Ship1 and Socs1 and altering TNF and IL‐10 production. In a murine model of LPS‐induced peritonitis, cold exposure potentiated hypothermia and decreased survival (10 vs. 50%; P < 0.05), effects that were associated with increased miR‐155, suppression of Ship1 and Socs1, and alterations in TNF and IL‐10. Importantly, miR‐155‐deficiency reduced hypothermia and improved survival (78 vs. 32%, P < 0.05), which was associated with increased Ship1, Socs1, and IL‐10. These results establish a causal role of miR‐155 in the dysregulation of the inflammatory response to hypothermia.— Billeter, A. T., Hellmann, J., Roberts, H., Druen, D., Gardner, S. A., Sarojini, H., Galandiuk, S., Chien, S., Bhatnagar, A., Spite, M., Polk, H. C., Jr. MicroRNA‐155 potentiates the inflammatory response in hypothermia by suppressing IL‐10 production. FASEB J. 28, 5322–5336 (2014). www.fasebj.org

Ischemic rabbit ear model created by minimally invasive surgery

A rabbit ear ischemic model was created using a minimally invasive surgical technique. On one ear, three small skin incisions were made on the vascular pedicles about 1u2003cm from the base of the ear. The central and cranial arteries were ligated and divided along with their accompanying nerves. A circumferential subcutaneous tunnel was made through the incisions to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a normal control. Wounds were made on the ventral side of the ear. Twenty‐two rabbits (14 young adults, four aged, and four diabetic) were used. In the 26 pairs of wounds in young adult rabbits, the mean healing time on the ischemic ear was 20.5±3.4 days vs. 14.9±1.6 days on the normal ear (mean±SD) when normal saline was used as a dressing. Tissue high‐energy phosphate contents were higher in the normal ear than in the ischemic ear. The skin temperature on the ischemic ear was 1–7u2003°C lower than that on the normal ear. Wound‐healing times were longer in the aged and diabetic rabbits, but no complications occurred in these rabbits. The model created by minimally invasive procedure results in little skin disruption, a longer ischemic time, and a higher success rate as compared with many other models. It can be used in normal animals as well as aged animals, and for the first time, was used successfully in diabetic animals.

Vaccinia virus complement control protein inhibits hyperacute xenorejection in a guinea pig-to-rat heterotopic cervical cardiac xenograft model by blocking both xenoantibody binding and complement pathway activation

Vaccinia virus complement control protein (VCP) binds the activated third and fourth complement components and inhibits both alternative and classical pathways of activation. The ability of VCP to bind heparan sulfate allows the protein to attach itself to the cell surface, enabling it with many additional activities. Altogether, the many functions of VCP have been shown to suppress the inflammatory response of the host, helping the vaccinia virus to evade immune destruction. VCP has recently been shown to inhibit human anti-Gal alpha1-3 Gal antibody attachment to cultured porcine endothelial cells and reduce human neutrophil and NK killing of pig aortic endothelial cells through its ability to bind heparan sulfate. Here we demonstrate that in an in vivo guinea pig-to-rat heterotopic cervical cardiac xenograft model, recombinant VCP (rVCP) is able to block hyperacute xenograft rejection, significantly prolonging graft survival. Histopathological examination of transplanted hearts from rats receiving rVCP revealed a significant reduction in cardiac tissue damage as compared to control hearts. Finally, rVCP treated recipients demonstrated marked rVCP deposition on the endothelium and significantly less C3, IgG and IgM deposition in the tissue. rVCP is therefore able to inhibit hyperacute xenorejection by binding the endothelial surface, blocking complement fixation and activation, and preventing xenoantibody attachment.

Rapid Granulation Tissue Regeneration by Intracellular ATP Delivery-A Comparison with Regranex

This study tests a new intracellular ATP delivery technique for tissue regeneration and compares its efficacy with that of Regranex. Twenty-seven adult New Zealand white rabbits each underwent minimally invasive surgery to render one ear ischemic. Eight wounds were then created: four on the ischemic and four on the normal ear. Two wounds on one side of each ear were treated with Mg-ATP encapsulated lipid vesicles (ATP-vesicles) while the two wounds on the other side were treated with Regranex. Wound healing time was shorter when ATP-vesicles were used. The most striking finding was that new tissue growth started to appear in less than 1 day when ATP-vesicles were used. The growth continued and covered the wound area within a few days, without the formation of a provisional matrix. Regranex-treated wounds did not have this growth pattern. In wounds treated by ATP-vesicles, histologic studies revealed extremely rich macrophage accumulation, along with active proliferating cell nuclear antigen (PCNA) and positive BrdU staining, indicating in situ macrophage proliferation. Human macrophage culture suggested direct collagen production. These results support an entirely new healing process, which seems to have combined the conventional hemostasis, inflammation, and proliferation phases into a single one, thereby eliminating the lag time usually seen during healing process.

The Vaccinia Virus N1L ORF May Encode a Multifunctional Protein Possibly Targeting Different Kinases, One of Which Influences ATP Levels in Vivo

As the single‐most potent virulence factor of the vaccinia virus, the 13.8‐kDa protein enhances viral replication in the brain by an unknown mechanism. Due to the high energy demands of the brain and the at times inadequate energy supply and small energy reserves to support physiologic activity, the ability of this organ to support energy requirements for replication of a virus is unlikely. We investigated the possible role of the 13.8‐kDa protein in the enhancement of adenosine triphosphate (ATP) utilization in the brain to sustain viral replication. In vitro and in vivo monitoring and comparison of ATP levels in mouse brain tissue infected with a wild‐type vaccinia virus or a 13.8‐kDa deletion strain (vGK5) revealed differences in ATP utilization and a significant difference in ATP levels in vivo after 5 days of infection. Because of poor replication of the wild‐type Lister vaccinia virus in the brain, a role for the 13.8‐kDa protein in the modulation of ATP levels to support viral replication in the brain could not be conclusively implicated. Evaluation of the amino acid sequence and predicted secondary structure of the 13.8‐kDa protein and sequence and structural homologs thereof provided evidence of putative dimerization and adenine binding sites and a possible kinase‐related function for this protein.

Pivotal role of ATP in macrophages fast tracking wound repair and regeneration

Chronic wounds occurring during aging or diabetes pose a significant burden to patients. The classical four‐phase wound healing process has a 3–6 day lag before granulation starts to appear and it requires an intermediate step of activation of resident fibroblasts during the remodeling phase for production of collagen. This brief communication discusses published articles that demonstrate how the entire wound healing process can be fast tracked by intracellular ATP delivery, which triggers a novel pathway where alternatively activated macrophages play absolutely critical and central roles. This novel pathway involves an increase in proinflammatory cytokines (TNF, IL‐1β, IL‐6) and a chemokine (MCP‐1) release. This is followed by activation of purinergic receptor (a family of plasma membrane receptors found in almost all mammalian cells), production of platelets and platelet microparticles, and activation of ATP‐dependent chromatin remodeling enzymes. The end result is a massive influx and in situ proliferation of macrophages, increases in vascular endothelial growth factors that promote neovascularization, and most prominently, the direct production of collagen.

Macrophage Differentiation in Normal and Accelerated Wound Healing

Chronic wounds pose considerable public health challenges and burden. Wound healing is known to require the participation of macrophages, but mechanisms remain unclear. The M1 phenotype macrophages have a known scavenger function, but they also play multiple roles in tissue repair and regeneration when they transition to an M2 phenotype. Macrophage precursors (mononuclear cells/monocytes) follow the influx of PMN neutrophils into a wound during the natural wound-healing process, to become the major cells in the wound. Natural wound-healing process is a four-phase progression consisting of hemostasis, inflammation, proliferation, and remodeling. A lag phase of 3-6xa0days precedes the remodeling phase, which is characterized by fibroblast activation and finally collagen production. This normal wound-healing process can be accelerated by the intracellular delivery of ATP to wound tissue. This novel ATP-mediated acceleration arises due to an alternative activation of the M1 to M2 transition (macrophage polarization), a central and critical feature of the wound-healing process. This response is also characterized by an early increased release of pro-inflammatory cytokines (TNF, IL-1 beta, IL-6), a chemokine (MCP-1), an activation of purinergic receptors (a family of plasma membrane receptors found in almost all mammalian cells), and an increased production of platelets and platelet microparticles. These factors trigger a massive influx of macrophages, as well as in situ proliferation of the resident macrophages and increased synthesis of VEGFs. These responses are followed, in turn, by rapid neovascularization and collagen production by the macrophages, resulting in wound covering with granulation tissue within 24xa0h.

Rapid tissue regeneration induced by intracellular ATP delivery—A preliminary mechanistic study

We have reported a new phenomenon in acute wound healing following the use of intracellular ATP delivery—extremely rapid tissue regeneration, which starts less than 24 h after surgery, and is accompanied by massive macrophage trafficking, in situ proliferation, and direct collagen production. This unusual process bypasses the formation of the traditional provisional extracellular matrix and significantly shortens the wound healing process. Although macrophages/monocytes are known to play a critical role in the initiation and progression of wound healing, their in situ proliferation and direct collagen production in wound healing have never been reported previously. We have explored these two very specific pathways during wound healing, while excluding confounding factors in the in vivo environment by analyzing wound samples and performing in vitro studies. The use of immunohistochemical studies enabled the detection of in situ macrophage proliferation in ATP-vesicle treated wounds. Primary human macrophages and Raw 264.7 cells were used for an in vitro study involving treatment with ATP vesicles, free Mg-ATP alone, lipid vesicles alone, Regranex, or culture medium. Collagen type 1α 1, MCP-1, IL-6, and IL-10 levels were determined by ELISA of the culture supernatant. The intracellular collagen type 1α1 localization was determined with immunocytochemistry. ATP-vesicle treated wounds showed high immunoreactivity towards BrdU and PCNA antigens, indicating in situ proliferation. Most of the cultured macrophages treated with ATP-vesicles maintained their classic phenotype and expressed high levels of collagen type 1α1 for a longer duration than was observed with cells treated with Regranex. These studies provide the first clear evidence of in situ macrophage proliferation and direct collagen production during wound healing. These findings provide part of the explanation for the extremely rapid tissue regeneration, and this treatment may hold promise for acute and chronic wound care.

Significant upregulation of U1 and U4 spliceosomal snRNAs by ATP nanoliposomes explains acceleration of wound healing, due to increased pre-mRNA processing to functional mRNA

Delayed wound healing is one of the hallmarks of diabetic complications and certain autoimmune inflammatory diseases. Extensive wound healing studies in rabbits have indicated that the delivery of ATP encapsulated in unilamellar nanoliposomes causes rapid cell proliferation and fast tracks the wound healing process. In the current study, we explored the possible molecular mechanism underlying this response by comparing gene expression in cultured rabbit kidney cells treated with either ATP nanoliposomes (containing 1 mg Mg-ATP/ml formulation) or control nanoliposomes (containing 1 mg/ml unmetabolisable gamma-thio-ATP/ml formulation). High-quality total RNA was isolated 24 h from the cells and subjected to RNA seq technology, which revealed significant overexpression of specific noncoding RNAs. The U1 spliceosomal RNA, U1 snRNA, was upregulated more than 250-fold following treatment with ATP nanoliposomes. This multifunctional U1 spliceosomal RNA may function in transcription by speeding up the critical splicing step, thereby facilitating faster processing of pre-mRNA to translatable mRNA.