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1.
Bone Joint Res ; 2(9): 186-92, 2013.
Article in English | MEDLINE | ID: mdl-24021530

ABSTRACT

OBJECTIVES: The goals of this study were: 1) to determine if high-fat diet (HFD) feeding in female mice would negatively impact biomechanical and histologic consequences on the Achilles tendon and quadriceps muscle; and 2) to investigate whether exercise and branched-chain amino acid (BCAA) supplementation would affect these parameters or attenuate any negative consequences resulting from HFD consumption. METHODS: We examined the effects of 16 weeks of 60% HFD feeding, voluntary exercise (free choice wheel running) and BCAA administration in female C57BL/6 mice. The Achilles tendons and quadriceps muscles were removed at the end of the experiment and assessed histologically and biomechanically. RESULTS: HFD feeding significantly decreased the Achilles tendon modulus without histological alterations. BCAA administration significantly decreased the stiffness of Achilles tendons in the exercised normal diet mice. Exercise partially ameliorated both the weight gain and glucose levels in the HFD-fed mice, led to a significant decrease in the maximum load of the Achilles tendon, and an increase in the average fibril diameter of the quadriceps femoris muscle. There were significant correlations between body weight and several biomechanical properties, demonstrating the importance of controlling obesity for maintaining healthy tendon properties. CONCLUSIONS: In summary, this study showed a significant impact of obesity and body weight on tendon biomechanical properties with limited effects of exercise and BCAAs. Cite this article: Bone Joint Res 2013;2:186-92.

2.
Oncogene ; 32(49): 5551-62, 2013 Dec 05.
Article in English | MEDLINE | ID: mdl-23752189

ABSTRACT

Breast cancer is one of the most common malignancies in human females in the world. One protein that has elevated enzymatic lipase activity in breast cancers in vitro is phospholipase D (PLD), which is also involved in cell migration. We demonstrate that the PLD2 isoform, which was analyzed directly in the tumors, is crucial for cell invasion that contributes critically to the growth and development of breast tumors and lung metastases in vivo. We used three complementary strategies in a SCID mouse model and also addressed the underlying molecular mechanism. First, the PLD2 gene was silenced in highly metastatic, aggressive breast cancer cells (MDA-MB-231) with lentivirus-based short hairpin RNA, which were xenotransplanted in SCID mice. The resulting mouse primary mammary tumors were reduced in size (65%, P<0.05) and their onset delayed when compared with control tumors. Second, we stably overexpressed PLD2 in low-invasive breast cancer cells (MCF-7) with a biscistronic MIEG retroviral vector and observed that these cells were converted into a highly aggressive phenotype, as primary tumors that formed following xenotransplantation were larger, grew faster and developed lung metastases more readily. Third, we implanted osmotic pumps into SCID xenotransplanted mice that delivered two different small-molecule inhibitors of PLD activity (5-fluoro-2-indolyl des-chlorohalopemide and N-[2-(4-oxo-1-phenyl-1,3,8-triazaspiro[4,5]dec-8-yl)ethyl]-2-naphthalenecarboxamide). These inhibitors led to significant (>70%, P<0.05) inhibition of primary tumor growth, metastatic axillary tumors and lung metastases. In order to define the underlying mechanism, we determined that the machinery of PLD-induced cell invasion is mediated by phosphatidic acid, Wiscott-Aldrich Syndrome protein, growth receptor-bound protein 2 and Rac2 signaling events that ultimately affect actin polymerization and cell invasion. In summary, this study shows for the first time that PLD2 has a central role in the development, metastasis and level of aggressiveness of breast cancer, raising the possibility that PLD2 could be used as a new therapeutic target.


Subject(s)
Breast Neoplasms/pathology , Phospholipase D/metabolism , Animals , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Movement , Cell Proliferation , Female , Humans , Lung Neoplasms/secondary , MCF-7 Cells , Mice , Mice, SCID , Neoplasm Invasiveness , Neoplasm Metastasis , Neoplasm Transplantation , Phosphatidic Acids/biosynthesis , Phospholipase D/antagonists & inhibitors , Phospholipase D/genetics , Protein Isoforms/metabolism , RNA Interference , RNA, Small Interfering , Signal Transduction
3.
Clin Exp Immunol ; 158(3): 317-24, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19747209

ABSTRACT

Transforming growth factor (TGF) beta1) is an immunoregulatory cytokine involved in self-tolerance and lymphocyte homeostasis. Tgfb1 knock-out (KO) mice develop severe multi-focal autoimmune inflammatory lesions due to [Ca(2+)]i deregulation in T cells, and die within 3 weeks after birth. Because the calcineurin inhibitor FK506 inhibits the hyperresponsiveness of Tgfb1(-/-) thymocytes, and because calcineurin Abeta (CNAbeta)-deficient mice do not reject allogenic tumours, we have generated Tgfb1(-/-) Cnab(-/-) mice to address whether CNAbeta deficiency prevents T cell activation and inflammation in Tgfb1(-/-) mice. Here we show that in Tgfb1(-/-) Cnab(-/-) mice inflammation is reduced significantly relative to that in Tgfb1(-/-) mice. However, both CD4(+) and CD8(+) T cells in double knock-out (DKO) mice are activated, as revealed by up-regulation of CD11a lymphocyte function-associated antigen-1 (LFA-1), CD44 and CD69 and down-regulation of CD62L. These data suggest that deficiency of CNAbeta decreases inflammatory lesions but does not prevent activation of autoreactive T cells. Also Tgfb1(-/-) T cells can undergo activation in the absence of CNAbeta, probably by using the other isoform of calcineurin (CNAalpha) in a compensatory manner. CNAbeta-deficient T cells undergo spontaneous activation in vivo and are activated upon anti-T cell receptor stimulation in vitro. Understanding the role of calcineurin in T cell regulation should open up new therapeutic opportunities for inflammation and cancer.


Subject(s)
Autoimmune Diseases/immunology , Calcineurin/deficiency , Inflammation/immunology , Transforming Growth Factor beta1/immunology , Animals , Autoimmune Diseases/pathology , Autoimmune Diseases/prevention & control , Calcineurin/immunology , Cell Differentiation/immunology , Cells, Cultured , Inflammation/pathology , Inflammation/prevention & control , Lymphocyte Activation/immunology , Lymphocyte Count , Mice , Mice, Knockout , Spleen/immunology , Survival Analysis , T-Lymphocyte Subsets/immunology
4.
J Biomech ; 39(12): 2205-12, 2006.
Article in English | MEDLINE | ID: mdl-16120443

ABSTRACT

By 2030, there will be 70 million people in the United States over the age of 65, and by 2050, 22% of the US population will be considered elderly. It is generally believed that injuries in the elderly heal slower and less completely than in adolescents or young adults. To evaluate aging effects on tissue repair a surgical injury was created in the middle third of one patellar tendon in 1- and 4-5-year-old New Zealand White rabbits. The biomechanical properties of the isolated repair tissues and contralateral normal tendon tissues were compared at 6, 12 and 26 weeks post-injury. We hypothesized that repair tissues would exhibit age-related reductions in biomechanical properties at all time intervals of healing, both based on raw data and when normalized to values from contralateral tendons. Repairs from both age groups were similar, with no significant increase in maximum stress, strain at maximum stress, or modulus between 6 and 12 weeks. At 26 weeks, the repairs in the 4-year-old rabbits had higher maximum stress values than repairs in the 1-year-old rabbits (p=0.03). There were no significant differences in the strain at maximum stress or modulus. When repair tissue properties were normalized to values in the contralateral normal tendon, the maximum stress of the patellar tendon repair tissue from the 4 year old was significantly greater than the corresponding value from the 1 year old at the 26 week time point (p=0.04). In conclusion, these findings do not support the presence of age-related declines in the biomechanics of healing tendon.


Subject(s)
Aging , Tendon Injuries/physiopathology , Tendons/physiopathology , Wound Healing , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Elasticity , Female , Humans , Male , Rabbits , Stress, Mechanical , Tensile Strength
5.
J Orthop Res ; 23(2): 287-93, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15734238

ABSTRACT

Successful tissue engineered repair in the aging adult requires an abundant source of autologous, multipotent mesenchymal stem cells (MSCs). Although the number of bone marrow-derived MSCs declines dramatically with aging, their effectiveness in repair with increasing age has not been studied. We tested the hypothesis that MSCs harvested from geriatric rabbits would not repair patellar tendon defects as well as MSCs harvested from younger adult rabbits. In a novel within-subjects experiment, autologous MSCs were isolated from 1-year old rabbits, culture expanded, and cryogenically preserved. After housing the rabbits for 3 years, MSCs were re-harvested from the 4-year old rabbits and expanded. Five hundred thousand thawed and fresh MSCs were each separately mixed with type I collagen gel (333.3 x 10(3) cells/mg collagen) 24 h before surgery, and the resulting constructs implanted in bilateral full-length central third tendon defects. Twelve weeks post-surgery, the bone-tendon repair-bone units were failed in tension. Intra-animal (paired) comparisons between repair tissue treated with 1-year old MSCs and repair tissue treated with 4-year old MSCs resulted in no significant differences (alpha=0.05) in material properties including maximum stress (10.8 MPa vs. 9.9 MPa; p=0.762), modulus (139.8 MPa vs. 146.2 MPa; p=0.914), and strain energy density (0.52 N mm/mm(3) vs. 0.53 N mm/mm(3); p=0.966). Despite an age-related trend, there were also no significant differences in structural properties including maximum force (62.9 N vs. 27.0 N; p=0.070), stiffness (24.9 N/mm vs. 12.0 N/mm; p=0.111), and strain energy (87.2 N mm vs. 31.4 N mm; p=0.061). A subset of the rabbits (n=4 1 yrMSC, n=2 4 yrMSC) showed the presence of ectopic bone in the repair region and were not included in the mechanical analyses. We conclude that in the rabbit model MSCs do not lose their benefit as a tendon repair therapy with aging and that MSCs can be cryogenically stored for 3 years and still effectively repair soft tissue injuries.


Subject(s)
Aging/physiology , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/physiology , Tendon Injuries/therapy , Animals , Biomechanical Phenomena , Female , Rabbits , Wound Healing
6.
J Orthop Res ; 22(5): 998-1003, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15304271

ABSTRACT

Mesenchymal stem cells (MSCs) have been used to repair connective tissue defects in several animal models. Compared to "natural healing" controls (no added cells), MSC-collagen gel constructs in rabbit tendon defects significantly improve repair biomechanics. However, ectopic bone forms in 28% of MSC-treated rabbit tendons. To understand the source of bone formation, three studies were performed. In the first study, the hypothesis was tested that MSCs delivered during surgery contribute to bone formation in the in vivo repair site. Adjacent histological sections in the MSC-treated repair tissue were examined for pre-labeled MSCs and for cells showing positive alkaline phosphatase (ALP) activity. Both cells were observed in serial sections in regions of ectopic bone. Contralateral "natural healing" tendons lacked both markers. In the other two studies, the effects of osteogenic supplements and construct geometry (monolayer vs. 3-D) on ALP activity were studied to test three hypotheses: that rabbit MSCs increase ALP activity over time in monolayer culture conditions; that adding osteogenic inducing supplements to the culture medium increases cellular protein in monolayer culture; and that rabbit MSCs increase ALP activity both in monolayer and in 3-D constructs, with and without media supplements. Culture in monolayer under similar conditions to in vivo (as in the first study) did not increase ALP at 2 or 4 weeks. Medium designed to increase osteogenic activity significantly increased cell numbers (cellular protein increased by 260%) but did not affect ALP activity either in monolayer or 3-D constructs (p>0.12). However, MSCs in 3-D constructs exhibited higher ALP activity than cells in monolayer, both in the presence (p<0.045) and absence of supplement (p<0.005). These results suggest that in vitro conditions may critically influence cell differentiation and protein expression. Mechanisms responsible for these effects are currently under investigation.


Subject(s)
Alkaline Phosphatase/metabolism , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/cytology , Osteogenesis , Tendons/surgery , Animals , Cell Differentiation , Female , Mesenchymal Stem Cells/enzymology , Rabbits
7.
J Orthop Res ; 20(6): 1315-22, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12472246

ABSTRACT

Injuries to soft tissues such as tendons are becoming ever more frequent among the elderly. While increasing levels of activity likely contribute to these injuries, age-related declines in tendon strength may also be important. Whether these declines in biomechanical properties are associated with changes in fibril diameter or collagen type remains in question. In this study, age-related changes were investigated in patellar tendons from young adult rabbits (1-year old, n = 17) and from rabbits at the onset of senescence (4-year old, n = 33). Patellar tendon biomechanics was correlated with both collagen fibril diameter and with the presence of type V collagen, a known regulator of collagen fibril diameter. We hypothesize that (a) aging from I to 4 years results in significant reductions in patellar tendon biomechanical properties, and (b) these age-related declines are associated with smaller fibril diameters and with the presence of type V collagen. Maximum stress declined 25% between I and 4 years of age (100.7 +/- 5.6 MPa and 74.3 +/- 3.4 MPa, respectively, p < 0.0003) (mean +/- SEM) and strain energy density declined 40% (p < 0.001). The distribution of collagen fibrils from 4-year old rabbits was skewed significantly towards smaller diameters compared to fibrils from 1-year old rabbits (p < 0.001). Type V collagen was observed only in the 4-year old rabbit tendons. These correlations suggest that with increasing age after skeletal maturity, type V collagen may help to regulate the assembly and thus diameter of collagen fibrils and thereby adversely affect patellar tendon strength.


Subject(s)
Aging/physiology , Patella/physiology , Tendons/physiology , Animals , Biomechanical Phenomena , Collagen Type V/analysis , Female , Rabbits , Tendons/chemistry , Tendons/pathology
8.
J Mol Cell Cardiol ; 33(10): 1815-28, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11603924

ABSTRACT

Tropomyosin, an essential component of the sarcomere, regulates muscle contraction through Ca(2+)-mediated activation. Familial hypertrophic cardiomyopathy (FHC) is caused by mutations in numerous cardiac sarcomeric proteins, including myosin heavy and light chains, actin, troponin T and I, myosin binding protein C, and alpha-tropomyosin. This study developed transgenic mouse lines that encode an FHC mutation in alpha-tropomyosin; this mutation is an amino acid substitution at codon 180 (Glu180Gly) which occurs in a troponin T binding region. Non-transgenic and control mice expressing wild-type alpha-tropomyosin demonstrate no morphological or physiological changes. Expression of exogenous mutant tropomyosin leads to a concomitant decrease in endogenous alpha-tropomyosin without altering the expression of other contractile proteins. Histological analysis shows that initial pathological changes, which include ventricular concentric hypertrophy, fibrosis and atrial enlargement, are detected within 1 month. The disease-associated changes progressively increase and result in death between 4 and 5 months. Physiological analyses of the FHC mice using echocardiography, work-performing heart analyses, and force measurements of cardiac myofibers, demonstrate dramatic functional differences in diastolic performance and increased sensitivity to calcium. This report demonstrates that mutations in alpha-tropomyosin can be severely disruptive of sarcomeric function, which consequently triggers a dramatic hypertrophic response that culminates in lethality.


Subject(s)
Cardiomyopathy, Hypertrophic, Familial/genetics , Mutation , Tropomyosin/genetics , Age Factors , Amino Acids/chemistry , Animals , Calcium/pharmacology , Dose-Response Relationship, Drug , Echocardiography , Mice , Mice, Transgenic , RNA/metabolism , Time Factors
9.
J Biol Chem ; 276(29): 26737-40, 2001 Jul 20.
Article in English | MEDLINE | ID: mdl-11389134

ABSTRACT

Mutations in the human ATP2A2 gene, encoding sarco(endo)plasmic reticulum Ca(2+)-ATPase isoform 2 (SERCA2), cause Darier disease, an autosomal dominant skin disease characterized by multiple keratotic papules in the seborrheic regions of the body. Mice with a single functional Atp2a2 allele (the mouse homolog of ATP2A2) were shown previously to have reduced levels of SERCA2 in heart and mildly impaired cardiac contractility and relaxation. Here we show that aged heterozygous mutant (Atp2a2(+/-)) mice develop squamous cell tumors of the forestomach, esophagus, oral mucosa, tongue, and skin. Squamous cell tumors occurred in 13/14 Atp2a2(+/-) mice but were not observed in age- and sex-matched wild-type controls. Hyperkeratinized squamous cell papillomas and carcinomas of the upper digestive tract were the most frequent finding among Atp2a2(+/-) mice, and many animals had multiple tumors. Western blot analyses showed that SERCA2 protein levels were reduced in skin and other affected tissues of heterozygous mice. The development of squamous cell tumors in aged Atp2a2(+/-) mice indicates that SERCA2 haploinsufficiency predisposes murine keratinocytes to neoplasia. These findings provide the first direct demonstration that a perturbation of Ca(2+) homeostasis or signaling can serve as a primary initiating event in cancer.


Subject(s)
Alleles , Calcium-Transporting ATPases/genetics , Carcinoma, Squamous Cell/genetics , Heterozygote , Animals , Carcinoma, Squamous Cell/enzymology , Mice , Papilloma/enzymology , Papilloma/genetics , Sarcoplasmic Reticulum Calcium-Transporting ATPases
10.
Arthritis Rheum ; 44(4): 794-801, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11315918

ABSTRACT

OBJECTIVE: Although increased vascularity was noted in early histopathologic studies of juvenile rheumatoid arthritis (JRA) synovium, the available data on angiogenesis in JRA are very limited. The main purposes of this study were to assess expression of the key angiogenic factors in JRA synovium, and to evaluate a SCID mouse model of JRA as an approach to study in vivo regulation of the expression of these factors in JRA. METHODS: RNase protection assay was used to assess the expression of the key angiogenic factors in fresh JRA synovium and in JRA synovial tissue fragments that had been minced and then implanted into SCID mice. Vascularity of the samples was assessed by immunohistochemical staining for von Willebrand factor. Synovial specimens obtained from patients with osteoarthritis (OA) or other noninflammatory arthropathies were used as controls. RESULTS: Detectable levels of messenger RNA for vascular endothelial growth factor and angiopoietin 1 and their respective receptors, as well as endoglin and thrombin receptors, were present in all JRA tissue specimens studied. The levels of expression of these factors in JRA tissues were significantly higher than those in tissues obtained from patients with OA or other noninflammatory arthropathies. Furthermore, increased expression of the key angiogenic factors in the fresh JRA tissues correlated with the exuberant revascularization of JRA minced tissue fragments implanted into SCID mice. This was in sharp contrast to the poor revascularization of implanted OA tissues. CONCLUSION: JRA synovium is characterized by high angiogenic activity. SCID mouse-human JRA synovium chimeras may provide a good approach to study the in vivo regulation of angiogenesis in JRA.


Subject(s)
Arthritis, Juvenile/metabolism , Endothelial Growth Factors/metabolism , Lymphokines/metabolism , Membrane Glycoproteins/metabolism , Neovascularization, Pathologic/metabolism , Synovial Membrane/metabolism , Angiopoietin-1 , Animals , Antigens, CD , Arthritis, Juvenile/immunology , Disease Models, Animal , Endoglin , Endothelial Growth Factors/genetics , Female , Humans , Lymphokines/genetics , Membrane Glycoproteins/genetics , Mice , Mice, Inbred BALB C , Mice, SCID , Osteoarthritis/metabolism , RNA, Messenger/metabolism , Receptors, Cell Surface , Receptors, Thrombin/genetics , Receptors, Thrombin/metabolism , Synovial Membrane/immunology , Synovial Membrane/transplantation , Tissue Transplantation , Transplantation, Heterologous , Vascular Cell Adhesion Molecule-1/genetics , Vascular Cell Adhesion Molecule-1/metabolism , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors
11.
Magn Reson Imaging ; 19(9): 1209-16, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11755731

ABSTRACT

The objective of this work was to determine the ability of magnetic resonance (MR) imaging with ultrasmall superparamagnetic iron oxide (USPIO) particles to provide quantitative measures of inflammation in autoimmune arthritis. Mice were injected intravenously or intra-articularly with USPIO followed by magnetic resonance and histological assessment of the knee joint. Comparisons were made between MR microimages and histology in naïve mice and mice with collagen-induced arthritis.Following intravenous administration, accumulation of USPIO was observed in the popliteal lymph nodes, but not the joint. Administration of USPIO intra-articularly resulted in signal loss in the joint. The MR signal intensity could be quantified and correlated with iron staining in the synovial lining. A marked increase in USPIO uptake and a corresponding decrease in signal intensity were observed in arthritic, compared to naïve mice. Areas of focal signal loss corresponded to foci of iron staining by histology. These studies may provide a basis for the clinical application of USPIO in arthritis for assessing disease severity and monitoring response to therapy.


Subject(s)
Arthritis, Infectious/diagnosis , Autoimmune Diseases/diagnosis , Contrast Media , Iron , Knee Joint/pathology , Magnetic Resonance Imaging/methods , Oxides , Animals , Arthritis, Infectious/pathology , Autoimmune Diseases/pathology , Dextrans , Ferrosoferric Oxide , Gadolinium DTPA , Knee Joint/immunology , Magnetite Nanoparticles , Male , Mice , Mice, Inbred DBA , Signal Processing, Computer-Assisted
12.
Comp Med ; 50(5): 511-5, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11099134

ABSTRACT

Mice lacking the NHE2 Na+/H+ gene develop gastritis of the glandular mucosa as early as the tenth day of life, achieving maximal intensity of inflammation from 17 to 19 days after birth and maximal atrophy at one year. We assessed the effects of this process in such mice to 16 months of age. The stomach of NHE2 null mutants was examined at 10, 17 to 20, 24 to 35 and 49 to 70 days, and at 12 to 16 months. The NHE2 wild-type (+/+) and NHE2 heterozygous (+/-) mice were compared with the NHE2 homozygous mutant mice (-/-). The stomach of the mutant mice at all ages was characterized by a substantially reduced number of parietal cells. The 10-day-old mouse stomach had a transmural infiltrate of primarily neutrophils. With increasing age, neutrophils were replaced by lymphocytes and plasma cells in the glandular mucosa of the mutant mice. Young adult 49- to 70-day-old mice had surface cell hyperplasia and expansion of the replicating cell population. Hyperplasia of enterochromaffin-like cells and antral gastrin cells accompanied profound fundic gland and surface cell hyperplasia, and became progressively more severe with increasing age of the NHE2-/- mice. Neoplasms were not found in the mutant or control mice. This gastritis differs from that of autoimmune gastritis in that it is transmural, begins in infancy, and is associated with a predominantly neutrophilic infiltrate in its early stages. Some of the histologic changes in the adult mice can be explained on the basis of prolonged achlorhydria. This mouse may be a suitable model for prolonged effects of achlorhydria.


Subject(s)
Gastric Mucosa/pathology , Gastritis, Atrophic/veterinary , Mice, Knockout/physiology , Rodent Diseases/pathology , Sodium-Hydrogen Exchangers/physiology , Stomach/pathology , Age Factors , Animals , Disease Models, Animal , Female , Gastritis, Atrophic/genetics , Gastritis, Atrophic/pathology , Immunohistochemistry/veterinary , Male , Mice , Rodent Diseases/genetics , Sodium-Hydrogen Exchangers/genetics
13.
Mol Ther ; 2(2): 147-52, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10947942

ABSTRACT

Treatments for rheumatoid arthritis and other inflammatory arthropathies are often ineffective at preventing joint destruction. Long-term genetic modification of the cells lining the joint space (synoviocytes) in vivo represents a potential method for the treatment of these chronic conditions. However, a vector capable of efficiently transducing synoviocytes in vivo for a persistent period has not been available. The present report describes the genetic modification of synoviocytes in vivo using recombinant adeno-associated virus. High-titer adeno-associated virus encoding the gene for Escherichia coli beta-galactosidase was injected into the knee joints of mice. Synovial tissues were then examined for beta-galactosidase transgene expression by in situ staining and by fluorometry. High-efficiency, persistent transgene expression was observed in the synovium with no evidence of vector-induced inflammation. Expression was observed for at least 7 months and was higher in arthritic than nonarthritic mice. Gene transfer of murine IL-4 to the joints of mice with collagen-induced arthritis led to detectable levels of IL-4 in the joint and protection from articular cartilage destruction. These data suggest that adeno-associated virus may be a useful vector for gene delivery to the synovium for the treatment of inflammatory arthropathies.


Subject(s)
Dependovirus/genetics , Gene Transfer Techniques , Genetic Therapy/methods , Interleukin-4/genetics , Synovial Membrane/metabolism , Animals , Arthritis/therapy , Cartilage, Articular/pathology , Chondrocytes/metabolism , Escherichia coli/enzymology , Genetic Vectors , Interleukin-4/therapeutic use , Male , Mice , Mice, Inbred DBA , Patella/metabolism , Proteoglycans/biosynthesis , Spectrometry, Fluorescence , Time Factors , Transgenes , beta-Galactosidase/metabolism
14.
J Immunol ; 165(3): 1557-63, 2000 Aug 01.
Article in English | MEDLINE | ID: mdl-10903764

ABSTRACT

IL-2 is generally considered a pro-inflammatory cytokine that exacerbates Th1-mediated disease states, such as autoimmune arthritis. Consistent with this role for IL-2, recent studies from our laboratory demonstrate that IL-2 mRNA is markedly increased during the acute stage of collagen-induced arthritis (CIA), an animal model of rheumatoid arthritis. To further define the role of IL-2 in CIA, the levels of IL-2 protein and its receptor and the effects of IL-2 administration were analyzed during CIA. IL-2 protein and IL-2R were preferentially expressed at disease onset, compared with later stages of disease. Administration of recombinant human IL-2 (rhIL-2) at, or just before, disease onset exacerbated disease; surprisingly, rhIL-2 given before disease onset inhibited CIA, associated with reduced cellular and humoral responses to type II collagen. Determination of in vivo serum levels of Th1 and Th2 cytokines in response to rhIL-2 treatment demonstrated that IFN-gamma, but not IL-4, was markedly up-regulated in response to IL-2. In mice treated with anti-IFN-gamma Ab, both early and late IL-2 administration exacerbated CIA. Thus, IL-2 can have two opposite effects on autoimmune arthritis, a direct stimulatory effect and an indirect suppressive effect that is mediated by IFN-gamma.


Subject(s)
Arthritis, Experimental/immunology , Collagen/immunology , Interleukin-2/immunology , Acute Disease , Animals , Arthritis, Experimental/etiology , Arthritis, Experimental/pathology , Arthritis, Experimental/prevention & control , B-Lymphocytes/immunology , Cattle , Chronic Disease , Humans , Immune Sera/administration & dosage , Immunosuppressive Agents/administration & dosage , Inflammation Mediators/administration & dosage , Injections, Intraperitoneal , Interferon-gamma/immunology , Interferon-gamma/physiology , Interleukin-2/administration & dosage , Interleukin-2/biosynthesis , Male , Mice , Mice, Inbred DBA , Receptors, Interleukin-2/biosynthesis , Recombinant Proteins/administration & dosage , T-Lymphocytes/immunology , Th1 Cells/immunology , Th1 Cells/metabolism
15.
J Biomed Mater Res ; 51(2): 233-40, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10825223

ABSTRACT

Mesenchymal stem cells (MSCs) were isolated from bone marrow, culture-expanded, and then seeded at 1, 4, and 8 million cells/mL onto collagen gel constructs designed to augment tendon repair in vivo. To investigate the effects of seeding density on the contraction kinetics and cellular morphology, the contraction of the cell/collagen constructs was monitored over time up to 72 h in culture conditions. Constructs seeded at 4 and 8 million cells/mL showed no significant differences in their gross appearance and dimensions throughout the contraction process. By contrast, constructs seeded at 1 million cells/mL initially contracted more slowly and their diameters at 72 h were 62 to 73% larger than those seeded at higher densities. During contraction, MSCs reoriented and elongated significantly with time. Implants prepared at higher seeding densities showed more well aligned and elongated cell nuclei after 72 h of contraction. Changes in nuclear morphology of the MSCs in response to physical constraints provided by the contracted collagen fibrils may trigger differentiation pathways toward the fibroblastic lineage and influence the cell synthetic activity. Controlling the contraction and organization of the cells and matrix will be critical for successfully creating tissue engineered grafts.


Subject(s)
Bone Marrow Cells/cytology , Collagen , Mesoderm/cytology , Tendon Injuries/therapy , Animals , Cell Culture Techniques/instrumentation , Cell Culture Techniques/methods , Cells, Cultured , Gels , Orthopedic Procedures , Rabbits
16.
J Orthop Res ; 18(1): 116-25, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10716287

ABSTRACT

This study tested the hypothesis that eliminating in vivo compression to the wrap-around, fibrocartilage-rich zone of the flexor digitorum profundus tendon results in rapid depletion of fibrocartilage and changes in its mechanical properties, microstructure, extracellular matrix composition, and cellularity. The right flexor digitorum profundus tendons of 2.5-3-year-old rabbits were translocated anteriorly to eliminate in vivo compression and shear to the fibrocartilage zone and, at 4 weeks after surgery, were compared with tendons that had sham surgery and with untreated tendons. The translocated tissue showed a significant increase in equilibrium strain under a compressive creep load (p < 0.05). The thickness and area of the fibrocartilage zone also decreased significantly (p < 0.05). The nuclear density decreased by 40% in the fibrocartilage zone (p < 0.005); however, nuclear shape and orientation were not significantly altered. Glycosaminoglycan content in the fibrocartilage zone was also depleted by 40% (p < 0.02). The tightly woven basket weave-like mesh of collagen fibers in the zone appeared more loosely organized, suggesting matrix reorganization due to translocation. Moreover, immunoreactive type-II collagen and link protein in the fibrocartilage zone also decreased. With use of this unique in vivo model, this research clearly elucidates how changing tissue function (by removing compressive forces) rapidly alters tissue form.


Subject(s)
Tendons/physiology , Animals , Biomechanical Phenomena , Cartilage/physiology , Collagen/analysis , Collagen/immunology , Glycosaminoglycans/analysis , Rabbits , Tendons/chemistry , Uronic Acids/analysis
17.
J Biol Chem ; 274(41): 29558-63, 1999 Oct 08.
Article in English | MEDLINE | ID: mdl-10506222

ABSTRACT

Tropomyosin is an essential component of the sarcomeric thin filament in striated muscle that participates in the regulation of muscle contraction through Ca(2+)-mediated activation. The two predominant tropomyosin isoforms expressed in striated muscle are alpha- and beta-tropomyosin, which exhibit an 86% amino acid identity between themselves. Previous studies by our laboratory utilized a transgenic mouse system to overexpress beta-tropomyosin in the heart to address the functional differences between these two tropomyosin isoforms. Interestingly, when a high percentage of beta-tropomyosin replaces alpha-tropomyosin in the hearts of transgenic mice, the mice die due to severe cardiac abnormalities. In this study, we have rescued these high expression beta-tropomyosin mice by turning off the alpha-myosin heavy chain promoter, which is driving the beta-tropomyosin transgene. This down-regulation of the alpha-myosin heavy chain promoter was accomplished by the administration of 5-propyl-2-thiouracil, which disrupts thyroid hormone synthesis and inhibits promoter activity through thyroid regulatory elements located in the 5'-flanking region of the promoter. Results show that as beta-tropomyosin expression is down-regulated, alpha-tropomyosin expression is increased. Also, alpha- and beta-myosin heavy chain expression is modified in response to the changes in thyroid hormone expression. Morphological analysis of these rescued mice show a moderate pathological phenotype, characterized by atrial myocytolysis; echocardiographic analyses demonstrate altered ventricular functions, such as peak filling rates and left ventricular fractional shortening. This is the first report demonstrating that transcriptional regulatory elements located within the alpha-myosin heavy chain promoter can be manipulated to rescue potentially lethal phenotypes, such as high expression beta-tropomyosin transgenic mice.


Subject(s)
Gene Expression Regulation/drug effects , Propylthiouracil/pharmacology , Tropomyosin/genetics , Animals , Down-Regulation/drug effects , Heart Atria/pathology , Heart Defects, Congenital/genetics , Heart Function Tests , Mice , Mice, Transgenic , Myosin Heavy Chains/genetics , Myosins/genetics , Organ Size , Phenotype , Promoter Regions, Genetic , RNA, Messenger/metabolism
18.
Tissue Eng ; 5(3): 267-77, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10434073

ABSTRACT

Mesenchymal stem cells (MSCs) were isolated from bone marrow of 18 adult New Zealand White rabbits. These cells were culture expanded, suspended in type I collagen gel, and implanted into a surgically induced defect in the donor s right patellar tendon. A cell-free collagen gel was implanted into an identical control defect in the left patellar tendon. Repair tissues were evaluated biomechanically (n = 13) and histomorphometrically (n = 5) at 4 weeks after surgery. Compared to their matched controls, the MSC-mediated repair tissue demonstrated significant increases of 26% (p < 0.001), 18% (p < 0. 01), and 33% (p < 0.02) in maximum stress, modulus, and strain energy density, respectively. Qualitatively, there appeared to be minor improvements in the histological appearance of some of the MSC- mediated repairs, including increased number of tenocytes and larger and more mature-looking collagen fiber bundles. Morphometrically, however, there were no significant left-right differences in nuclear aspect ratio (shape) or nuclear alignment (orientation). Therefore, delivering a large number of mesenchymal stem cells to a wound site can significantly improve its biomechanical properties by only 4 weeks but produce no visible improvement in its microstructure.


Subject(s)
Cell Transplantation , Mesoderm/cytology , Stem Cells/cytology , Tendon Injuries/therapy , Animals , Biomechanical Phenomena , Bone Marrow Cells/cytology , Cell Culture Techniques/methods , Cell Division , Cells, Cultured , Patella/injuries , Rabbits , Tendon Injuries/physiopathology , Transplantation, Autologous
19.
Cancer Res ; 59(14): 3379-86, 1999 Jul 15.
Article in English | MEDLINE | ID: mdl-10416598

ABSTRACT

The transforming growth factor beta (TGF-beta) pathway is known to play an important role in both human and urine colon cancer. However, the staging, ligand specificity, and mechanism underlying the tumor suppressive activity of this pathway are unknown. We developed a mouse model for colon cancer that identifies an early role for TGF-beta1 in tumor suppression and implicates TGF-beta2 or TGF-beta3 in the prevention of metastasis. Analysis of the development of colon cancer in TGF-beta1 knockout mice pinpoints the defect to the hyperplasty/adenoma transition and reveals that the mechanism involves an inability to maintain epithelial tissue organization and not a loss of growth control, increased inflammatory activity, or increased genetic instability. These mice provide a unique opportunity to investigate the specific role of TGF-beta1 at this critical transition in the development of colon cancer.


Subject(s)
Colonic Neoplasms/drug therapy , Trans-Activators , Transforming Growth Factor beta/therapeutic use , Adenocarcinoma/drug therapy , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Adenoma/drug therapy , Adenoma/genetics , Adenoma/pathology , Adenomatous Polyposis Coli Protein , Animals , Apoptosis , Biomarkers , Cecum/enzymology , Cecum/pathology , Cell Division , Cell Transformation, Neoplastic/drug effects , Colon/enzymology , Colon/pathology , Colonic Neoplasms/pathology , Crosses, Genetic , Cytoskeletal Proteins/analysis , DNA/genetics , DNA, Neoplasm/genetics , DNA-Binding Proteins , Disease Progression , Genes, APC , Genetic Predisposition to Disease , Humans , Hyperplasia , Inflammation/enzymology , Intestinal Mucosa/pathology , Mice , Mice, Knockout , Microsatellite Repeats , Neoplasm Metastasis , Nuclear Proteins , Specific Pathogen-Free Organisms , Transforming Growth Factor beta/deficiency , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/physiology , beta Catenin
20.
Circ Res ; 85(1): 47-56, 1999 Jul 09.
Article in English | MEDLINE | ID: mdl-10400910

ABSTRACT

To investigate the functional consequences of a tropomyosin (TM) mutation associated with familial hypertrophic cardiomyopathy (FHC), we generated transgenic mice that express mutant alpha-TM in the adult heart. The missense mutation, which results in the substitution of asparagine for aspartic acid at amino acid position 175, occurs in a troponin T binding region of TM. S1 nuclease mapping and Western blot analyses demonstrate that increased expression of the alpha-TM 175 transgene in different lines causes a concomitant decrease in levels of endogenous alpha-TM mRNA and protein expression. In vivo physiological analyses show a severe impairment of both contractility and relaxation in hearts of the FHC mice, with a significant change in left ventricular fractional shortening. Myofilaments that contain alpha-TM 175 demonstrate an increased activation of the thin filament through enhanced Ca2+ sensitivity of steady-state force. Histological analyses show patchy areas of mild ventricular myocyte disorganization and hypertrophy, with occasional thrombi formation in the left atria. Thus, the FHC alpha-TM transgenic mouse can serve as a model system for the examination of pathological and physiological alterations imparted through aberrant TM isoforms.


Subject(s)
Cardiomyopathy, Hypertrophic/genetics , Cardiomyopathy, Hypertrophic/physiopathology , Heart/physiopathology , Mutation/physiology , Tropomyosin/genetics , Animals , Calcium/physiology , Cardiomyopathy, Hypertrophic/pathology , Homeostasis/physiology , Mice , Mice, Transgenic/genetics , Myocardial Contraction/physiology , Myocardium/pathology
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