miR-125b regulates chemotaxis and survival of bone marrow derived granulocytes in vitro and in vivo.

The evolutionary conserved miR-125b is highly expressed in hematopoietic stem cells (HSC) enhancing self-renewal and survival. Accordingly, over-expression of miR-125b in HSC may induce myeloproliferative neoplasms and leukemia with long latency. During hematopoietic cell maturation miR-125b expression decreases, and the function of miR-125b in mature granulocytes is not yet known. We here use transplantation of miR-125b over-expressing HSC into syngeneic hosts to generate and analyse miR-125b over-expressing granulocytes. Under steady state conditions, miR-125b over-expression inhibits granulocytic chemotaxis and LPS- but not PMA- and TNFα- induced cell death. Inflammatory signals modulate the effects of miR-125b over-expression as demonstrated in a sterile peritonitis and a polymicrobial sepsis model. In particular, survival of mice with miR-125b over-expressing granulocytes is significantly reduced as compared to controls in the polymicrobial sepsis model. These data demonstrate inflammation dependent effects of miR-125b in granulocytes and may point to therapeutic intervention strategies in the future.

[Trying to unravel an unresolved issue in regenerative medicine: gene expression profiling of MSCs]. / Genexpressionsmuster mesenchymaler Stammzellen: ein ungelöster Aspekt in der regenerativen Medizin.

BACKGROUND: Mesenchymal stem cells (MSCs) are adult fibroblastoid progenitor cells. Because of their immunoregulatory properties and their so-called trophic effects, MSCs play an important role in tissue regeneration, inflammation and trauma. Tissue trauma and challenge, for example during radiotherapy or infection, result in the release of so-called "danger molecules", which may be derived from dying cells or incoming pathogens. The molecular response of MSCs to this tissue stress remains largely elusive. MATERIAL AND METHODS: In this study we examined the cell biological response of MSCs derived from human parotid glands (pgMSCs) and used bacterial endotoxin as a model of tissue stress and inflammation. PgMSCs from 3 donors were isolated, expanded and tested for classical tri-lineage plus myogenic differentiation. The cell biological response to the model "stressor" endotoxin was examined by low density gene expression arrays. RESULTS: Through immunofluorescence and immunohistochemistry we were able to proof osteogenic, adipogenic, chondrogenic, and myogenic differentiation potential characteristic for stem cells. In vitro, gene expression analysis showed a characteristic modulation of MSCs after stimulation with endotoxin Lipopolysaccharide (LPS). Specifically, receptors and ligands typically involved in immune regulation, such as interleukins, TGF-ß, tumor necrosis factors (TNF), and toll-like receptors (TLR), were regulated. CONCLUSION: Our study elucidates some key functions and molecules, which are regulated in MSCs during tissue stress and inflammation. A thorough understanding of their cell biological function will aid future rationale therapeutic application of MSCs.

Hepatocyte growth factor, a determinant of airspace homeostasis in the murine lung.

The alveolar compartment, the fundamental gas exchange unit in the lung, is critical for tissue oxygenation and viability. We explored hepatocyte growth factor (HGF), a pleiotrophic cytokine that promotes epithelial proliferation, morphogenesis, migration, and resistance to apoptosis, as a candidate mediator of alveolar formation and regeneration. Mice deficient in the expression of the HGF receptor Met in lung epithelial cells demonstrated impaired airspace formation marked by a reduction in alveolar epithelial cell abundance and survival, truncation of the pulmonary vascular bed, and enhanced oxidative stress. Administration of recombinant HGF to tight-skin mice, an established genetic emphysema model, attenuated airspace enlargement and reduced oxidative stress. Repair in the TSK/+ mouse was punctuated by enhanced akt and stat3 activation. HGF treatment of an alveolar epithelial cell line not only induced proliferation and scattering of the cells but also conferred protection against staurosporine-induced apoptosis, properties critical for alveolar septation. HGF promoted cell survival was attenuated by akt inhibition. Primary alveolar epithelial cells treated with HGF showed improved survival and enhanced antioxidant production. In conclusion, using both loss-of-function and gain-of-function maneuvers, we show that HGF signaling is necessary for alveolar homeostasis in the developing lung and that augmentation of HGF signaling can improve airspace morphology in murine emphysema. Our studies converge on prosurvival signaling and antioxidant protection as critical pathways in HGF-mediated airspace maintenance or repair. These findings support the exploration of HGF signaling enhancement for diseases of the airspace.

Calpain 1 knockdown improves tissue sparing and functional outcomes after spinal cord injury in rats.

To evaluate the hypothesis that calpain 1 knockdown would reduce pathological damage and functional deficits after spinal cord injury (SCI), we developed lentiviral vectors encoding calpain 1 shRNA and eGFP as a reporter (LV-CAPN1 shRNA). The ability of LV-CAPN1 shRNA to knockdown calpain 1 was confirmed in rat NRK cells using Northern and Western blot analysis. To investigate the effects on spinal cord injury, LV-CAPN1shRNA or LV-mismatch control shRNA (LV-control shRNA) were administered by convection enhanced diffusion at spinal cord level T10 in Long-Evans female rats (200-250 g) 1 week before contusion SCI, 180 kdyn force, or sham surgery at the same thoracic level. Intraspinal administration of the lentiviral particles resulted in transgene expression, visualized by eGFP, in spinal tissue at 2 weeks after infection. Calpain 1 protein levels were reduced by 54% at T10 2 weeks after shRNA-mediated knockdown (p<0.05, compared with the LV-control group, n=3 per group) while calpain 2 levels were unchanged. Intraspinal administration of LV-CAPN1shRNA 1 week before contusion SCI resulted in a significant improvement in locomotor function over 6 weeks postinjury, compared with LV-control administration (p<0.05, n=10 per group). Histological analysis of spinal cord sections indicated that pre-injury intraspinal administration of LV-CAPN1shRNA significantly reduced spinal lesion volume and improved total tissue sparing, white matter sparing, and gray matter sparing (p<0.05, n=10 per group). Together, results support the hypothesis that calpain 1 activation contributes to the tissue damage and impaired locomotor function after SCI, and that calpain1 represents a potential therapeutic target.

Viral gene transfer to developing mouse salivary glands.

Branching morphogenesis is essential for the formation of salivary glands, kidneys, lungs, and many other organs during development, but the mechanisms underlying this process are not adequately understood. Microarray and other gene expression methods have been powerful approaches for identifying candidate genes that potentially regulate branching morphogenesis. However, functional validation of the proposed roles for these genes has been severely hampered by the absence of efficient techniques to genetically manipulate cells within embryonic organs. Using ex vivo cultured embryonic mouse submandibular glands (SMGs) as models to study branching morphogenesis, we have identified new vectors for viral gene transfer with high efficiency and cell-type specificity to developing SMGs. We screened adenovirus, lentivirus, and 11 types of adeno-associated viruses (AAV) for their ability to transduce embryonic day 12 or 13 SMGs. We identified two AAV types, AAV2 and bovine AAV (BAAV), that are selective in targeting expression differentially to SMG epithelial and mesenchymal cell populations, respectively. Transduction of SMG epithelia with self-complementary (sc) AAV2 expressing fibroblast growth factor 7 (Fgf7) supported gland survival and enhanced SMG branching morphogenesis. Our findings represent, to our knowledge, the first successful selective gene targeting to epithelial vs. mesenchymal cells in an organ undergoing branching morphogenesis.

Tumor necrosis factor-a antisense transfer remarkably improves hepatic graft viability.

BACKGROUND: Cold ischemia/reperfusion injury of the hepatic graft, an unsolved problem in liver transplantations, is attributed to the release of inflammatory cytokines, especially the tumor necrosis factor- (TNF) alpha, from activated Kupffer cells (KC). Therefore, the specific inhibition of TNF-alpha could improve the viability of the hepatic graft upon reperfusion. METHODS: We assessed the efficacy of TNF-alpha antisense (TNF-AS) oligodeoxynucleotides (ODNs) delivery to KC in a rodent liver transplantation model. RESULTS: Seventy-one percent of the animals that received 6 hours preserved grafts in baths of lactated Ringer's solution (4 degrees C) and were treated with TNF-AS survived for over 14 days. Eighty percent of the animals treated with vehicle, sense ODNs, or balanced salt saline (BSS) died. Four hours after reperfusion of the liver, a significant reduction was noted in livers treated with TNF-AS in the release of cytosolic enzymes from the hepatocytes and the serum TNF-alpha (P<0.05). The expressions of TNF-alpha on KC and of intercellular adhesion molecule-1 on sinusoidal endothelial cells were completely suppressed in TNF-AS-treated livers. CONCLUSIONS: TNF-AS delivery improves the viability of the hepatic graft, and this technique may solve hepatic graft nonfunction in a clinical setting.

[Angiogenesis VEGF165 gene therapy with AdVEGF- a new delay procedure for flaps]. / Angiogenese-Gentherapie mit AdVEGF.

A regular tissue functioning requires the adequate supply of oxygen and nutrient via blood vessels. The sequences of formation and maturation of vessels are initiated and maintained by different growth factors. The VEGF growth factor plays an exceptional role in these mechanisms. The creation of sublethal ischemia as an angiogenic stimulus known as "Delay" is a well established procedure in plastic surgery, although the underlying molecular biological mechanisms still remain unknown. The important role of VEGF and its regulation depending on oxygen pressure suggest a strong connection between this growth factor and the delay phenomenon. The VEGF concentration in skin and underlying muscle was measured in overdimensioned random pattern flaps on 32 male Sprague-Dawley rats after either VEGF gene therapy or circumcision without elevation of the flap and compared to controls. Additional random pattern flaps were raised seven days post gene therapy or delay. The effect on the flap perfusion was measured postoperatively using Indocyanine green Laser Fluoroscopy and the size of the surviving and necrotic areas of the flaps were analysed. The skin of the random pattern flaps showed both in the Delay group and in the VEGF gene therapy group a significantly elevated VEGF concentration compared to the controls. The underlying rectus abdominis muscle showed no significant differences in VEGF concentration between the groups. The flap perfusion postoperatively was significantly increased solely in the VEGF gene therapy group. The analysis of the surviving area of the flaps showed a significant increase over the controls in the gene therapy group. The Delay procedure results in a significantly and locally raised concentration of the VEGF growth factor. The gene therapeutical use of this growth factor allows us to raise flap perfusion and to reduce necrosis. Both VEGF gene therapy and Delay seem to promote similar mechanisms whereas the gene therapy produced superior results in this setting.

Gene transfer as a tool to induce therapeutic vascular growth in plastic surgery.

Sufficient tissue blood perfusion is critical for wound healing and for reconstructions in plastic surgery. There is considerable interest in the use of therapeutic angiogenesis to improve survival of ischemic flaps and to improve wound healing. Several growth factors have been tested in experimental flap models. Clinically, angiogenic therapy has been extensively studied in cardiovascular ischemic diseases and several clinical studies are ongoing in human patients. Results from these cardiovascular trials will benefit the planning of clinical trials in the field of plastic surgery. Furthermore, the development of strategies for local and controlled induction of lymphangiogenesis will hopefully improve the treatment of edema.

Retrospective molecular detection of Transthyretin Met 111 mutation in a Danish kindred with familial amyloid cardiomyopathy, using DNA from formalin-fixed and paraffin-embedded tissues.

Severe familial amyloid cardiomyopathy (FAC) in a Danish kindred is associated with a specific mutation (Met for Leu 111) in the transthyretin (TTR) gene. The mutation causes the loss of a DdeI restriction site in the gene, allowing molecular diagnostic studies. We studied formalin-fixed, paraffin-embedded tissues, up to 39 years old, from 29 family members of this kindred. DNA was partially purified from deparaffinized tissue sections and a DNA sequence of the TTR gene flanking the mutation site was amplified by the polymerase chain reaction (PCR), followed by restriction enzyme analysis. Amplified DNA was obtained from tissues representing 23 of the 29 persons. Ten out of the 23 family members were found to carry the TTR Met 111 mutation, whereas 13 were not affected. The results were consistent with known clinical data and with corresponding serum TTR examinations. This retrospective study shows that archival tissues can be used to confirm the diagnosis and disease pattern in members of families affected by hereditary diseases.