Equine bone marrow MSC-derived extracellular vesicles mitigate the inflammatory effects of interleukin-1ß on navicular tissues in vitro.

BACKGROUND: Safe, efficacious therapy for treating degenerate deep digital flexor tendon (DDFT) and navicular bone fibrocartilage (NBF) in navicular horses is critically necessary. While archetypal orthobiologic therapies for navicular disease are used empirically, their safety and efficacy are unknown. Mesenchymal stem cell-derived extracellular vesicles (EV) may overcome several limitations of current orthobiologic therapies. OBJECTIVES: To (1) characterise cytokine and growth factor profiles of equine bone marrow mesenchymal stem cell (BM-MSC)-derived extracellular vesicles (BM-EV) and (2) evaluate the in vitro anti-inflammatory and extracellular matrix (ECM) protective potentials of BM-EV on DDFT and NBF explant co-cultures in an IL-1ß inflammatory environment. STUDY DESIGN: In vitro experimental study. METHODS: Cytokines (IL-1ß, IL-6, IL-10, IL-1ra and TNF-α) and growth factors (TGFß1, VEGF, IGF1 and PDGF) in equine BM-EV isolated via ultracentrifugation and precipitation methods were profiled. Forelimb DDFT and NBF explant co-cultures from seven horses were exposed to media alone, or media containing 2 × 109 ± 0.1 × 109 particles/mL or 10 µg/mL BM-EV (BM-EV), 10 ng/mL interleukin-1ß (IL-1ß), or IL-1ß + BM-EV for 48 h. Co-culture media IL-6, TNF-α, MMP-3, MMP-13 concentrations and explant sulphated glycosaminoglycan (sGAG) content were quantified. RESULTS: IL-6, IGF1 and VEGF concentrations were 102.1 (37.61-256.2) and 182.3 (163.1-226.3), 72.3 (8-175.6) and 2.4 (0.1-2.6), 108.3 (38.3-709.1) and 211.4 (189.1-318.2) pg/mL per 2 × 109 ± 0.1 × 109 particles/mL or 10 µg/mL 10 µg of BM-EV isolated via ultracentrifugation and precipitation methods, respectively. Co-culture media MMP-3 in BM-EV- (p = 0.03) and BM-EV + IL-1ß-treated (p = 0.01) groups were significantly lower than the respective media and IL-1ß groups. DDFT explant sGAG content of BM-EV (p = 0.003) and BM-EV + IL-1ß groups were significantly higher compared with IL-1ß group. MAIN LIMITATIONS: Specimen numbers are limited, in vitro model may not replicate clinical case conditions, lack of non-MSC-derived EV control group. CONCLUSIONS: Equine BM-EV contains IL-6 and growth factors, IGF1 and VEGF. The anti-inflammatory and ECM protective potentials of BM-EV were evident as increased IL-6 and decreased MMP-3 concentrations in the DDFT-NBF explant co-culture media. These results support further evaluation of BM-EV as an acellular and 'off-the-shelf' intra-bursal/intrasynovial therapy for navicular pathologies.

Xenogenic Implantation of Human Mesenchymal Stromal Cells Using a Novel 3D-Printed Scaffold of PLGA and Graphene Leads to a Significant Increase in Bone Mineralization in a Rat Segmental Femoral Bone Defect.

Tissue-engineering technologies have the potential to provide an effective approach to bone regeneration. Based on the published literature and data from our laboratory, two biomaterial inks containing PLGA and blended with graphene nanoparticles were fabricated. The biomaterial inks consisted of two forms of commercially available PLGA with varying ratios of LA:GA (65:35 and 75:25) and molecular weights of 30,000-107,000. Each of these forms of PLGA was blended with a form containing a 50:50 ratio of LA:GA, resulting in ratios of 50:65 and 50:75, which were subsequently mixed with a 0.05 wt% low-oxygen-functionalized derivative of graphene. Scanning electron microscopy showed interconnected pores in the lattice structures of each scaffold. The cytocompatibility of human ADMSCs transduced with a red fluorescent protein (RFP) was evaluated in vitro. The in vivo biocompatibility and the potential to repair bones were evaluated in a critically sized 5 mm mechanical load-bearing segmental femur defect model in rats. Bone repair was monitored by radiological, histological, and microcomputed tomography methods. The results showed that all of the constructs were biocompatible and did not exhibit any adverse effects. The constructs containing PLGA (50:75)/graphene alone and with hADMSCs demonstrated a significant increase in mineralized tissues within 60 days post-treatment. The percentage of bone volume to total volume from microCT analyses in the rats treated with the PLGA + cells construct showed a 50% new tissue formation, which matched that of a phantom. The microCT results were supported by Von Kossa staining.

A pilot study to demonstrate the paracrine effect of equine, adult allogenic mesenchymal stem cells in vitro, with a potential for healing of experimentally-created, equine thoracic wounds in vivo.

Regenerative biological therapies using mesenchymal stem cells (MSCs) are being studied and used extensively in equine veterinary medicine. One of the important properties of MSCs is the cells' reparative effect, which is brought about by paracrine signaling, which results in the release of biologically active molecules, which in turn, can affect cellular migration and proliferation, thus a huge potential in wound healing. The objective of the current study was to demonstrate the in vitro and in vivo potentials of equine allogenic bone marrow-derived MSCs for wound healing. Equine bone marrow-derived MSCs from one allogenic donor horse were used. Equine MSCs were previously characterized for their in vitro proliferation, expression of cluster-of-differentiation markers, and trilineage differentiation. MSCs were first evaluated for their migration using an in vitro wound healing scratch assay, and subsequently, the conditioned medium was evaluated for their effect on human fibroblast proliferation. Subsequently, allogenic cells were intradermally injected into full-thickness, cutaneous thoracic wounds of 4 horses. Wound healing was assessed by using 3-D digital imaging and by measuring mRNA expression of pro-and anti-inflammatory markers for 30 days. Using human fibroblasts in an in vitro wound healing assay, we demonstrate a significantly higher healing in the presence of conditioned medium collected from proliferating MSCs than in the presence of medium containing fetal bovine serum. The in vitro effect of MSCs did not translate into a detectable effect in vivo. Nonetheless, we proved that molecularly characterized equine allogenic MSCs do not illicit an immunologic response. Investigations using MSCs derived from other sources (adipose tissue, umbilical cord), or a higher number of MSCs or a compromised animal model may be required to prove the efficacy of equine MSCs in wound healing in vivo.

3D-Printing Graphene Scaffolds for Bone Tissue Engineering.

Graphene-based materials have recently gained attention for regenerating various tissue defects including bone, nerve, cartilage, and muscle. Even though the potential of graphene-based biomaterials has been realized in tissue engineering, there are significantly many more studies reporting in vitro and in vivo data in bone tissue engineering. Graphene constructs have mainly been studied as two-dimensional (2D) substrates when biological organs are within a three-dimensional (3D) environment. Therefore, developing 3D graphene scaffolds is the next clinical standard, yet most have been fabricated as foams which limit control of consistent morphology and porosity. To overcome this issue, 3D-printing technology is revolutionizing tissue engineering, due to its speed, accuracy, reproducibility, and overall ability to personalize treatment whereby scaffolds are printed to the exact dimensions of a tissue defect. Even though various 3D-printing techniques are available, practical applications of 3D-printed graphene scaffolds are still limited. This can be attributed to variations associated with fabrication of graphene derivatives, leading to variations in cell response. This review summarizes selected works describing the different fabrication techniques for 3D scaffolds, the novelty of graphene materials, and the use of 3D-printed scaffolds of graphene-based nanoparticles for bone tissue engineering.

Muscle Regeneration of the Tongue: A Review of Current Clinical and Regenerative Research Strategies.

Various abnormalities of the tongue, including cancers, commonly require surgical removal to sequester growth and metastasis. However, even minor resections can affect functional outcomes such as speech and swallowing, thereby reducing quality of life. Surgical resections alone create volumetric muscle loss whereby muscle tissue cannot self-regenerate within the tongue. In these cases, the tongue is reconstructed typically in the form of autologous skin flaps. However, flap reconstruction has many limitations and unfortunately is the primary option for oral and reconstructive surgeons to treat tongue defects. The alternative, but yet undeveloped, strategy for tongue reconstruction is regenerative medicine, which widely focuses on building new organs with stem cells. Regenerative medicine has successfully treated many tissues, but research has inadequately addressed the tongue as a vital organ in need of tissue engineering. In this review, we address the current standard for tongue reconstruction, the cellular mechanisms of muscle cell development, and the stem cell studies that have attempted muscle engineering within the tongue. Until now, no review has focused on engineering the tongue with regenerative medicine, which could guide innovative strategies for tongue reconstruction. Impact statement Unlike other bodily organs, the current literature has inadequately addressed the tongue as a vital organ in need of tissue engineering. Therefore, this review seeks to highlight the clinical challenges of tongue reconstruction, alternative tissue engineering strategies, and to summarize the studies involving muscle regeneration within the tongue. This information will guide maxillofacial surgeons and tissue engineering scientists to pursue innovative strategies that alleviate volumetric muscle loss in the tongue.

Etched 3D-Printed Polycaprolactone Constructs Functionalized with Reduced Graphene Oxide for Enhanced Attachment of Dental Pulp-Derived Stem Cells.

A core challenge in the field of tissue engineering is the ability to establish pipeline workflows for the design and characterization of scaffold technologies with clinically translatable attributes. The parallel development of biomaterials and stem cell populations represents a self-sufficient and streamlined approach for establishing such a pipeline. In the current study, rat dental pulp stem cell (rDPSC) populations were established to assess functionalized polycaprolactone (PCL) constructs. Initial optimization and characterization of rDPSC extraction and culture conditions confirmed that cell populations were readily expandable and demonstrated surface markers associated with multi-potency. Subset populations were transduced to express DsRed fluorescent protein as a mechanism of tracking both cells and cell-derived extracellular matrix content on complex scaffold architecture. Thermoplastic constructs included reduced graphene oxide (rGO) as an additive to promote cellular attachment and were further modified by surface etching a weak acetic acid solution to roughen surface topographical features, which was observed to dramatically improve cell surface coverage in vitro. Based on these data, the modified rGO-functionalized PCL constructs represent a versatile platform for bone tissue engineering, capable of being applied as a standalone matrix or in conjunction with bio-active payloads such as DPSCs or other bio-inks.

Genetic profiling of human bone marrow and adipose tissue-derived mesenchymal stem cells reveals differences in osteogenic signaling mediated by graphene.

BACKGROUND: In the last decade, graphene surfaces have consistently supported osteoblast development of stem cells, holding promise as a therapeutic implant for degenerative bone diseases. However, until now no study has specifically examined the genetic changes when stem cells undergo osteogenic differentiation on graphene. RESULTS: In this study, we provide a detailed overview of gene expressions when human mesenchymal stem cells (MSCs) derived from either adipose tissue (AD-MSCs) or bone marrow (BM-MSCs), are cultured on graphene. Genetic expressions were measured using osteogenic RT2 profiler PCR arrays and compared either over time (7 or 21 days) or between each cell source at each time point. Genes were categorized as either transcriptional regulation, osteoblast-related, extracellular matrix, cellular adhesion, BMP and SMAD signaling, growth factors, or angiogenic factors. Results showed that both MSC sources cultured on low oxygen graphene surfaces achieved osteogenesis by 21 days and expressed specific osteoblast markers. However, each MSC source cultured on graphene did have genetically different responses. When compared between each other, we found that genes of BM-MSCs were robustly expressed, and more noticeable after 7 days of culturing, suggesting BM-MSCs initiate osteogenesis at an earlier time point than AD-MSCs on graphene. Additionally, we found upregulated angiogenic markers in both MSCs sources, suggesting graphene could simultaneously attract the ingrowth of blood vessels in vivo. Finally, we identified several novel targets, including distal-less homeobox 5 (DLX5) and phosphate-regulating endopeptidase homolog, X-linked (PHEX). CONCLUSIONS: Overall, this study shows that graphene genetically supports differentiation of both AD-MSCs and BM-MSCs but may involve different signaling mechanisms to achieve osteogenesis. Data further demonstrates the lack of aberrant signaling due to cell-graphene interaction, strengthening the application of specific form and concentration of graphene nanoparticles in bone tissue engineering.

Bone and Cartilage Interfaces With Orthopedic Implants: A Literature Review.

The interface between a surgical implant and tissue consists of a complex and dynamic environment characterized by mechanical and biological interactions between the implant and surrounding tissue. The implantation process leads to injury which needs to heal over time and the rapidity of this process as well as the property of restored tissue impact directly the strength of the interface. Bleeding is the first and most relevant step of the healing process because blood provides growth factors and cellular material necessary for tissue repair. Integration of the implants placed in poorly vascularized tissue such as articular cartilage is, therefore, more challenging than compared with the implants placed in well-vascularized tissues such as bone. Bleeding is followed by the establishment of a provisional matrix that is gradually transformed into the native tissue. The ultimate goal of implantation is to obtain a complete integration between the implant and tissue resulting in long-term stability. The stability of the implant has been defined as primary (mechanical) and secondary (biological integration) stability. Successful integration of an implant within the tissue depends on both stabilities and is vital for short- and long-term surgical outcomes. Advances in research aim to improve implant integration resulting in enhanced implant and tissue interface. Numerous methods have been employed to improve the process of modifying both stability types. This review provides a comprehensive discussion of current knowledge regarding implant-tissue interfaces within bone and cartilage as well as novel approaches to strengthen the implant-tissue interface. Furthermore, it gives an insight into the current state-of-art biomechanical testing of the stability of the implants. Current knowledge reveals that the design of the implants closely mimicking the native structure is more likely to become well integrated. The literature provides however several other techniques such as coating with a bioactive compound that will stimulate the integration and successful outcome for the patient.

Functionalized gold nanorod nanocomposite system to modulate differentiation of human mesenchymal stem cells into neural-like progenitors.

A 2D multifunctional nanocomposite system of gold nanorods (AuNRs) was developed. Gold nanorods were functionalized via polyethylene glycol with a terminal amine, and, were characterized using transmission and scanning electron microscopy, ultra violet-visible and X-ray photoelectron spectroscopy, and Zeta-potential. The system was cytocompatible to and maintained the integrity of Schwann cells. The neurogenic potential of adipose tissue - derived human mesenchymal stem cells (hMSCs) was evaluated in vitro. The expression pattern and localization of Vimentin confirmed the mesenchymal origin of cells and tracked morphological changes during differentiation. The expression patterns of S100ß and glial fibrillary acidic protein (GFAP), were used as indicator for neural differentiation. Results suggested that this process was enhanced when the cells were seeded on the AuNRs compared to the tissue-culture surface. The present study indicates that the design and the surface properties of the AuNRs enhances neural differentiation of hMSCs and hence, would be beneficial for neural tissue engineering scaffolds.

A Novel Model for Acute Peripheral Nerve Injury in the Horse and Evaluation of the Effect of Mesenchymal Stromal Cells Applied In Situ on Nerve Regeneration: A Preliminary Study.

Transplantation of mesenchymal stromal cells (MSCs) to sites of experimentally created nerve injury in laboratory animals has shown promising results in restoring nerve function. This approach for nerve regeneration has not been reported in horses. In this study, we first evaluated the in vitro ability of equine bone marrow-derived MSCs (EBM-MSCs) to trans-differentiate into Schwann-like cells and subsequently tested the MSCs in vivo for their potential to regenerate a transected nerve after implantation. The EBM-MSCs from three equine donors were differentiated into SCLs for 7 days, in vitro, in the presence of specialized differentiation medium and evaluated for morphological characteristics, by using confocal microscopy, and for protein characteristics, by using selected Schwann cell markers (GFAP and S100b). The EBM-MSCs were then implanted into the fascia surrounding the ramus communicans of one fore limb of three healthy horses after a portion of this nerve was excised. The excised portion of the nerve was examined histologically at the time of transection, and stumps of the nerve were examined histologically at day 45 after transplantation. The EBM-MSCs from all donors demonstrated morphological and protein characteristics of those of Schwann cells 7 days after differentiation. Nerves implanted with EBM-MSCs after nerve transection did not show evidence of nerve regeneration at day 45. Examination of peripheral nerves collected 45 days after injury and stem cell treatment revealed no histological differences between nerves treated with MSCs and those treated with isotonic saline solution (controls). The optimal delivery of MSCs and the model suitable to study the efficacy of MSCs in nerve regeneration should be investigated.

Donor-Matched Comparison of Chondrogenic Potential of Equine Bone Marrow- and Synovial Fluid-Derived Mesenchymal Stem Cells: Implications for Cartilage Tissue Regeneration.

Mesenchymal stem cells (MSCs) have been demonstrated to be useful for cartilage tissue regeneration. Bone marrow (BM) and synovial fluid (SF) are promising sources for MSCs to be used in cartilage regeneration. In order to improve the clinical outcomes, it is recommended that prior to clinical use, the cellular properties and, specifically, their chondrogenic potential must be investigated. The purpose of this study is to compare and better understand the in vitro chondrogenic potential of equine bone marrow-derived mesenchymal stem cells (BMMSCs) and synovial fluid-derived mesenchymal stem cells (SFMSCs) populated from the same equine donor. BM- and SF-derived MSCs cultures were generated from five equine donors, and the MSCs were evaluated in vitro for their morphology, proliferation, trilineage differentiation, and immunophenotyping. Differences in their chondrogenic potentials were further evaluated quantitatively using glycosaminoglycan (GAG) content and via immunofluorescence of chondrogenic differentiation protein markers, SRY-type HMG box9, Aggrecan, and collagen II. The BMMSCs and SFMSCs were similar in cellular morphology, viability, and immunophenotype, but, varied in their chondrogenic potential, and expression of the key chondrogenic proteins. The SFMSCs exhibited a significant increase in GAG content compared to the BMMSCs (P < 0.0001) in three donors, suggesting increased levels of chondrogenesis. The expression of the key chondrogenic proteins correlated positively with the GAG content, suggesting that the differentiation process is dependent on the expression of the target proteins in these three donors. Our findings suggest that even though SFMSCs were hypothesized to be more chondrogenic relative to BMMSCs, there was considerable donor-to-donor variation in the primary cultures of MSCs which can significantly affect their downstream application.

Bone-tissue engineering: complex tunable structural and biological responses to injury, drug delivery, and cell-based therapies.

Bone loss and failure of proper bone healing continues to be a significant medical condition in need of solutions that can be implemented successfully both in human and veterinary medicine. This is particularly true when large segmental defects are present, the bone has failed to return to normal form or function, or the healing process is extremely prolonged. Given the inherent complexity of bone tissue - its unique structural, mechanical, and compositional properties, as well as its ability to support various cells - it is difficult to find ideal candidate materials that could be used as the foundation for tissue regeneration from technological platforms. Recently, important developments have been made in the implementation of complex structures built both at the macro- and the nano-level that have been shown to positively impact bone formation and to have the ability to deliver active biological molecules (drugs, growth factors, proteins, cells) for controlled tissue regeneration and the prevention of infection. These materials are diverse, ranging from polymers to ceramics and various composites. This review presents developments in this area with a focus on the role of scaffold structure and chemistry on the biologic processes that influence bone physiology and regeneration.

Cell proliferation, viability, and in vitro differentiation of equine mesenchymal stem cells seeded on bacterial cellulose hydrogel scaffolds.

The culture of multipotent mesenchymal stem cells on natural biopolymers holds great promise for treatments of connective tissue disorders such as osteoarthritis. The safety and performance of such therapies relies on the systematic in vitro evaluation of the developed stem cell-biomaterial constructs prior to in vivo implantation. This study evaluates bacterial cellulose (BC), a biocompatible natural polymer, as a scaffold for equine-derived bone marrow mesenchymal stem cells (EqMSCs) for application in bone and cartilage tissue engineering. An equine model was chosen due to similarities in size, load and types of joint injuries suffered by horses and humans. Lyophilized and critical point dried BC hydrogel scaffolds were characterized using scanning electron microscopy (SEM) to confirm nanostructure morphology which demonstrated that critical point drying induces fibre bundling unlike lyophilisation. EqMSCs positively expressed the undifferentiated pluripotent mesenchymal stem cell surface markers CD44 and CD90. The BC scaffolds were shown to be cytocompatible, supporting cellular adhesion and proliferation, and allowed for osteogenic and chondrogenic differentiation of EqMSCs. The cells seeded on the BC hydrogel were shown to be viable and metabolically active. These findings demonstrate that the combination of a BC hydrogel and EqMSCs are promising constructs for musculoskeletal tissue engineering applications.

mRNA expression of canine ATP10C, a P4-type ATPase, is positively associated with body condition score.

Mouse and human Atp10c genes are strong candidates for changes in bodyweight and glucose homeostasis. Using comparative genomic analysis, a novel canine P4-type ATPase, ATP10C, was identified. Expression of ATP10C was compared between sex-matched lean (body condition score, BCS<8; n=7) and obese (BCS⩾8, n=8) client-owned dogs of comparable ages. Canine ATP10C is highly expressed in visceral and subcutaneous fat at approximately 3-fold levels compared to the omental adipose depot. There was a 5-fold significant increase (P<0.0001) in mRNA expression of ATP10C in dogs with a BCS⩾8.

Age-dependent regulation of sodium-potassium adenosinetriphosphatase and sodium-hydrogen exchanger mRNAs in equine nonglandular mucosa.

OBJECTIVE: To determine whether expression of mRNA for sodium-potassium adenosine-triphosphatase (NAKA) and sodium-hydrogen exchanger (NHE) in samples of the nonglandular portion of the equine gastric mucosa was altered by exposure to volatile fatty acids (VFAs) in an acidic environment. ANIMALS: 10 horses (5 < or = 5 years old and 5 > or = 12 years old). PROCEDURES: Samples of the nonglandular portion of the gastric mucosa were collected and exposed in Ussing chambers to Ringer's solution (control samples), Ringer's solution containing a mixture of VFAs (pH, 1.5 or 4.0), or Ringer's solution containing acetic acid (pH, 1.5 or 4.0). Expression of mRNA for the gene for the beta1 subunit of NAKA and the gene for the NHE-3 isoform was determined by means of real-time PCR assays. RESULTS: For horses < or = 5 years old, relative expression of mRNA for NAKA was significantly decreased and expression of mRNA for NHE was significantly increased following exposure to the mixture of VFAs or acetic acid, compared with expression in control samples. In contrast, for horses > or = 12 years old, relative expression of mRNA for both NAKA and NHE was significantly increased following exposure to the mixture of VFAs or acetic acid, compared with expression in control samples. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that relative expression of mRNA for NAKA, but not NHE, in samples of the nonglandular portion of the equine gastric mucosa in response to exposure to VFAs in an acidic environment was an age-dependent event.

G-Protein Inwardly Rectifying Potassium Channel 1 (GIRK1) Knockdown Decreases Beta-Adrenergic, MAP Kinase and Akt Signaling in the MDA-MB-453 Breast Cancer Cell Line.

Previous data from our laboratory have indicated that there is a functional link between the beta-adrenergic receptor signaling pathway and the G-protein inwardly rectifying potassium channel (GIRK1) in breast cancer cell lines and that these pathways are involved in growth regulation of these cells. To determine functionality, MDA-MB-453 breast cancer cells were stimulated with ethanol, known to open GIRK channels. Decreased GIRK1 protein levels were seen after treatment with 0.12% ethanol. In addition, serum-free media completely inhibited GIRK1 protein expression. This data indicates that there are functional GIRK channels in breast cancer cells and that these channels are involved in cellular signaling. In the present research, to further define the signaling pathways involved, we performed RNA interference (siRNA) studies. Three stealth siRNA constructs were made starting at bases 1104, 1315, and 1490 of the GIRK1 sequence. These constructs were transfected into MDA-MB-453 cells, and both RNA and protein were isolated. GIRK1, ß(2)-adrenergic and 18S control levels were determined using real-time PCR 24 hours after transfection. All three constructs decreased GIRK1 mRNA levels. However, ß(2) mRNA levels were unchanged by the GIRK1 knockdown. GIRK1 protein levels were also reduced by the knockdown, and this knockdown led to decreases in beta-adrenergic, MAP kinase and Akt signaling.

Protein expression of G-protein inwardly rectifying potassium channels (GIRK) in breast cancer cells.

BACKGROUND: Previous data from our laboratory has indicated that a functional link exists between the G-protein-coupled inwardly rectifying potassium (GIRK) channel and the beta-adrenergic receptor pathway in breast cancer cell lines, and these pathways were involved in growth regulation of these cells. Alcohol is an established risk factor for breast cancer and has been found to open GIRK. In order to further investigate GIRK channels in breast cancer and possible alteration by ethanol, we identified GIRK channel protein expression in breast cancer cells. RESULTS: Cell pellets were collected and membrane protein was isolated to determine GIRK protein expression. GIRK protein was also analyzed by immuno-precipitation. GIRK protein was over-expressed in cells by transfection of GIRK plasmids. Gene expression studies were done by real-time RT-PCR. GIRK protein expression was identified in breast cancer cell lines. Expression of GIRK1 at the indicated molecular weight (MW) (62 kDa) was seen in cell lines MDA-MB-453 and ZR-75-1. In addition, GIRK1 expression was seen at a lower MW (40-42 kDa) in MDA-MB-361, MDA-MB-468, MCF-7, ZR-75-1, and MDA-MB-453 cell lines. To prove the lower MW protein was GIRK1, MDA-MB-453 cells were immuno-precipitated. GIRK2 expression was seen in MDA-MB-468, MCF-7, and ZR-75-1 and was variable in MDA-MB-453, while GIRK4 protein expression was seen in all six cell lines tested. This is the first report indicating GIRK protein expression in breast cancer cells. To determine functionality, MDA-MB-453 cells were stimulated with ethanol. Decreased GIRK1 protein expression levels were seen after treatment with 0.12% ethanol in MDA-MB-453 breast cancer cells. Serum-free media decreased GIRK protein expression, possibly due to lack of estrogen in the media. Transfection of GIRK1 or GIRK4 plasmids increased GIRK1 protein expression and decreased gene expression in MDA-MB-453 breast cancer cells. CONCLUSION: Our data indicates that functional GIRK channels exist in breast cancer cells that are involved in cellular signaling.

A type IV P-type ATPase affects insulin-mediated glucose uptake in adipose tissue and skeletal muscle in mice.

Mice carrying two pink-eyed dilution (p) locus heterozygous deletions represent a novel polygenic mouse model of type 2 diabetes associated with obesity. Atp10c, a putative aminophospholipid transporter on mouse chromosome 7, is a candidate for the phenotype. The phenotype is diet-induced. As a next logical step in the validation and characterization of the model, experiments to analyze metabolic abnormalities associated with these mice were carried out. Results demonstrate that mutants (inheriting the p deletion maternally) heterozygous for Atp10c are hyperinsulinemic, insulin-resistant and have an altered insulin-stimulated response in peripheral tissues. Adipose tissue and the skeletal muscle are the targets, and GLUT4-mediated glucose uptake is the specific metabolic pathway associated with Atp10c deletion. Insulin resistance primarily affects the adipose tissue and the skeletal muscle, and the effect in the liver is secondary. Gene expression profiling using microarray and real-time PCR show significant changes in the expression of four genes--Vamp2, Dok1, Glut4 and Mapk14--involved in insulin signaling. The expression of Atp10c is also significantly altered in the adipose tissue and the soleus muscle. The most striking observation is the loss of Atp10c expression in the mutants, specifically in the soleus muscle, after eating the high-fat diet for 12 weeks. In conclusion, experiments suggest that the target genes and/or their cognate factors in conjunction with Atp10c presumably affect the normal translocation and sequestration of GLUT4 in both the target tissues.

Expression of G-protein inwardly rectifying potassium channels (GIRKs) in lung cancer cell lines.

BACKGROUND: Previous data from our laboratory has indicated that there is a functional link between the beta-adrenergic receptor signaling pathway and the G-protein inwardly rectifying potassium channel (GIRK1) in human breast cancer cell lines. We wanted to determine if GIRK channels were expressed in lung cancers and if a similar link exists in lung cancer. METHODS: GIRK1-4 expression and levels were determined by reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. GIRK protein levels were determined by western blots and cell proliferation was determined by a 5-bromo-2'-deoxyuridine (BrdU) assay. RESULTS: GIRK1 mRNA was expressed in three of six small cell lung cancer (SCLC) cell lines, and either GIRK2, 3 or 4 mRNA expression was detected in all six SCLC cell lines. Treatment of NCI-H69 with beta2-adrenergic antagonist ICI 118,551 (100 microM) daily for seven days led to slight decreases of GIRK1 mRNA expression levels. Treatment of NCI-H69 with the beta-adrenergic agonist isoproterenol (10 microM) decreased growth rates in these cells. The GIRK inhibitor U50488H (2 microM) also inhibited proliferation, and this decrease was potentiated by isoproterenol. In the SCLC cell lines that demonstrated GIRK1 mRNA expression, we also saw GIRK1 protein expression. We feel these may be important regulatory pathways since no expression of mRNA of the GIRK channels (1 & 2) was found in hamster pulmonary neuroendocrine cells, a suggested cell of origin for SCLC, nor was GIRK1 or 2 expression found in human small airway epithelial cells. GIRK (1,2,3,4) mRNA expression was also seen in A549 adenocarcinoma and NCI-H727 carcinoid cell lines. GIRK1 mRNA expression was not found in tissue samples from adenocarcinoma or squamous cancer patients, nor was it found in NCI-H322 or NCI-H441 adenocarcinoma cell lines. GIRK (1,3,4) mRNA expression was seen in three squamous cell lines, GIRK2 was only expressed in one squamous cell line. However, GIRK1 protein expression was not seen in any non-SCLC cells. CONCLUSION: We feel that this data may indicate that stimulation of GIRK1 or GIRK2 channels may be important in lung cancer. Stimulation of GIRK channels and beta-adrenergic signaling may activate similar signaling pathways in both SCLC and breast cancer, but lead to different results.

Lack of Pwcr1/MBII-85 snoRNA is critical for neonatal lethality in Prader-Willi syndrome mouse models.

Prader-Willi syndrome (PWS) is a neurobehavioral disorder caused by the lack of paternal expression of imprinted genes in the human chromosome region 15q11-13. Recent studies of rare human translocation patients narrowed the PWS critical genes to a 121-kb region containing PWCR1/HBII-85 and HBII-438 snoRNA genes. The existing mouse models of PWS that lack the expression of multiple genes, including Snrpn, Ube3a, and many intronic snoRNA genes, are characterized by 80%-100% neonatal lethality. To define the candidate region for PWS-like phenotypes in mice, we analyzed the expression of several genetic elements in mice carrying the large radiation-induced p(30PUb) deletion that includes the p locus. Mice having inherited this deletion from either parent develop normally into adulthood. By Northern blot and RT-PCR assays of brain tissue, we found that Pwcr1/MBII-85 snoRNAs are expressed normally, while MBII-52 snoRNAs are not expressed when the deletion is paternally inherited. Mapping of the distal deletion breakpoint indicated that the p30PUb deletion includes the entire MBII-52 snoRNA gene cluster and three previously unmapped EST sequences. The lack of expression of these elements in mice with a paternal p30PUb deletion indicates that they are not critical for the neonatal lethality observed in PWS mouse models. In addition, we identified MBII-436, the mouse homolog of the HBII-436 snoRNA, confirmed its imprinting status, and mapped it outside of the p30PUb deletion. Taking together all available data, we conclude that the lack of Pwcr1/MBII-85 snoRNA expression is the most likely cause for the neonatal lethality in PWS model mice.