Insight into the development of obesity: functional alterations of adipose-derived mesenchymal stem cells.

Obesity is associated with a variety of disorders including cardiovascular diseases, diabetes mellitus and cancer. Obesity changes the composition and structure of adipose tissue, linked to pro-inflammatory environment, endocrine/metabolic dysfunction, insulin resistance and oxidative stress. Adipose-derived mesenchymal stem cells (ASCs) have multiple functions like cell renewal, spontaneous repair and homeostasis in adipose tissue. In this review article, we have summarized the recent data highlighting that ASCs in obesity are defective in various functionalities and properties including differentiation, angiogenesis, motility, multipotent state, metabolism and immunomodulation. Inflammatory milieu, hypoxia and abnormal metabolites in obese tissue are crucial for impairing the functions of ASCs. Further work is required to explore the precise molecular mechanisms underlying its alterations and impairments. Based on these data, we suggest that deregulated ASCs, possibly also other mesenchymal stem cells, are important in promoting the development of obesity. Restoration of ASCs/mesenchymal stem cells might be an additional strategy to combat obesity and its associated diseases.

Deficiency of RITA results in multiple mitotic defects by affecting microtubule dynamics.

Deregulation of mitotic microtubule (MT) dynamics results in defective spindle assembly and chromosome missegregation, leading further to chromosome instability, a hallmark of tumor cells. RBP-J interacting and tubulin-associated protein (RITA) has been identified as a negative regulator of the Notch signaling pathway. Intriguingly, deregulated RITA is involved in primary hepatocellular carcinoma and other malignant entities. We were interested in the potential molecular mechanisms behind its involvement. We show here that RITA binds to tubulin and localizes to various mitotic MT structures. RITA coats MTs and affects their structures in vitro as well as in vivo. Tumor cell lines deficient of RITA display increased acetylated α-tubulin, enhanced MT stability and reduced MT dynamics, accompanied by multiple mitotic defects, including chromosome misalignment and segregation errors. Re-expression of wild-type RITA, but not RITA Δtub ineffectively binding to tubulin, restores the phenotypes, suggesting that the role of RITA in MT modulation is mediated via its interaction with tubulin. Mechanistically, RITA interacts with tubulin/histone deacetylase 6 (HDAC6) and its suppression decreases the binding of the deacetylase HDAC6 to tubulin/MTs. Furthermore, the mitotic defects and increased MT stability are also observed in RITA-/- mouse embryonic fibroblasts. RITA has thus a novel role in modulating MT dynamics and its deregulation results in erroneous chromosome segregation, one of the major reasons for chromosome instability in tumor cells.

Less understood issues: p21(Cip1) in mitosis and its therapeutic potential.

p21(Cip1) is a multifunctional protein and a key player in regulating different cellular processes. The transcription of p21 is regulated by p53-dependent and -independent pathways. The expression of p21 is increased in response to various cellular stresses to arrest the cell cycle and ensure genomic stability. p21 has been shown to be a tumor suppressor and an oncogene as well. The function of p21 in mitosis has been proposed but not systematically studied. We have recently shown that p21 binds to and inhibits the activity of Cdk1/cyclin B1, and is important for a fine-tuned mitotic progression. Loss of p21 prolongs the duration of mitosis and results in severe mitotic defects like chromosome segregation and cytokinesis failures promoting consequently genomic instability. Moreover, p21 is dramatically stabilized in mitotic tumor cells upon treatment with mitotic agents like paclitaxel or mitotic kinase inhibitors. Increased p21 is mainly localized in the cytoplasm and associates with cell survival indicating a crucial role of p21 in susceptibility to mitotic agents in tumor cells. In this review we will briefly summarize the structure and general physiological functions as well as regulation of p21, discuss in detail its role in mitosis and its potential to serve as a therapeutic target.

p21Waf1/Cip1 deficiency causes multiple mitotic defects in tumor cells.

As a multifaceted molecule, p21 plays multiple critical roles in cell cycle regulation, differentiation, apoptosis, DNA repair, senescence, aging and stem cell reprogramming. The important roles of p21 in the interphase of the cell cycle have been intensively investigated. The function of p21 in mitosis has been proposed but not systematically studied. We show here that p21 is abundant in mitosis and binds to and inhibits the activity of Cdk1/cyclin B1. Deficiency of p21 prolongs the duration of mitosis by extending metaphase, anaphase and cytokinesis. The activity of Aurora B is reduced and the localization of Aurora B on the central spindle is disturbed in anaphase cells without p21. Moreover, HCT116 p21-/-, HeLa and Saos-2 cells depleted of p21 encounter problems in chromosome segregation and cytokinesis. Gently inhibiting the mitotic Cdk1 or add-back of p21 rescues segregation defect in HCT116 p21-/- cells. Our data demonstrate that p21 is important for a fine-tuned control of the Cdk1 activity in mitosis, and its proper function facilitates a smooth mitotic progression. Given that p21 is downregulated in the majority of tumors, either by the loss of tumor suppressors like p53 or by hyperactive oncogenes such as c-myc, this finding also sheds new light on the molecular mechanisms by which p21 functions as a tumor suppressor.

Restoration of the tumor suppressor p53 by downregulating cyclin B1 in human papillomavirus 16/18-infected cancer cells.

Abrogation of functional p53 is responsible for malignant cell transformation and the maintenance of malignant state of human papillomavirus-infected cancer cells. Thus, restoration of p53 has been regarded as an important strategy for molecular intervention combating papillomavirus-associated malignancies. We show here that depleting cyclin B1 stabilizes and reactivates p53 in papillomavirus-infected cervical cancer cell lines HeLa and CaSki. HeLa cells depleted of cyclin B1 exhibit mitotic defects in spindle formation and chromosome alignment. Downregulation of cyclin B1 increases p14 alternative reading frame of p16, the positive regulator of p53, and decreases phosphorylation of Ser315 in p53. Whereas RO-3306, a selective inhibitor of cyclin-dependent kinase 1 (Cdk1), suppresses this phosphorylation at Ser315 of p53, ZM447439, targeting Aurora A/B kinases, shows no effect. Further analyses in HeLa cells and HCT116 p53(-/-) cells suggest that the Ser315 phosphorylation by Cdk1 regulates negatively the protein stability and the function of p53. Moreover, increased p53 in HeLa cells is functional by showing its increased downstream effectors p21, mouse double minute 2 and Bax. Restoration of p53 and silencing cyclin B1 render cervical carcinoma cells more susceptible to DNA damage agent camptothecin. Taken together, targeting cyclin B1 might be an attractive strategy for preventing and treating papillomavirus-associated cancer by reactivating p53 and by reducing the Cdk1 activity.

Leg symptoms of healthy people and their treatment with compression hosiery.

OBJECTIVES: Occasional leg symptoms, like feelings of heaviness and tension, and occupational or evening oedema are considered typical features of a venous disorder but show low specificity in epidemiological and observational studies. We evaluated the prevalence and nature of such symptoms in subjects with no history or signs of venous disease and investigated the optimal strength that medical compression stockings (MCS) should exert in order to alleviate the symptoms and to prevent leg swelling. METHODS: Specifically designed questionnaires were used to assess the symptoms of 40 healthy employees of a factory producing MCS. Lower leg volumes were quantified in the morning and evening. Calf size hosiery providing documented ankle pressures of 4-9 (mean 7.3), 12-18 (mean 14.9) and 18-22 (mean 19.5) mmHg, respectively, were tested in a prospective, open-label, randomized trial lasting three weeks. Endpoints were the relief of symptoms, prevention of vesperal oedema and comfort in wearing the stockings. RESULTS: Sixty-five percent of the participants reported at least occasional leg symptoms and oedema. Somatic-type symptoms (i.e. pain, heaviness, swelling, unattractive legs) were present in two, psychic-type symptoms (i.e. leg- and personality-related unrest and stress) in 17 and both components in seven of the 40 subjects. MCS exerting 15 and 20 mmHg prevented the symptoms and oedema. Stockings providing <10 mmHg were ineffective and those providing >19 mmHg were not well-tolerated. The effect on the somatic-type symptoms was strongly correlated with the amount of lower leg volume which could be reduced by wearing stockings (P = 0.005). No correlation was found between the efficacy of compression and the emotional component of the symptoms. CONCLUSION: The cause of occasional pain in the legs of apparently healthy people is unknown. Some features of the syndrome reflect an emotional disorder while others mirror venous insufficiency. MCS of 15 mmHg effectively relieve the symptoms resembling venous insufficiency, prevent oedema and are well-tolerated.

Practicability and quality of outpatient management of acute deep venous thrombosis.

PURPOSE: The purpose of this study was to review the practicability and quality of a standardized management approach of deep venous thrombosis (DVT) provided by private practices. METHODS: There were 152 consecutive patients and 156 episodes. We determined the patients' diagnoses with estimation of clinical probability, D-dimers, duplex ultrasound scan, and venography. Patients were treated on an outpatient basis on principle, with dalteparin, phenprocoumon, different modalities of external leg compression, and deliberate ambulation. We followed up at 4 weeks. RESULTS: Proximal DVT was diagnosed in 101 episodes (65%). Results of the D-dimer test were false-negative in 6%, and venography was indicated in 15%. Calf vein thrombosis was found in 55 patients. Results of the D-dimer test were false-negative in 30%, and venography was required in 37%. Eleven patients were hospitalized (9 for thrombectomy or thrombolysis), and 145 episodes (93%) were treated according to our standardized approach (provided by the referring physicians alone in 43%). For 5 days, dalteparin was injected by the patients themselves or their relatives, in 78% of the cases. The international normalized ratio values were more than 2 in 88% of the cases, with no difference between providers. In all but two cases, external leg compression was applied immediately: a modified Unna's boot in 28% and compressing stockings in 72% (Sigvaris 503 in 91%; calf length in 100% of distal DVT, and 83% of proximal DVT). During follow-up, there was no clinical evidence of recurrence or progression, 1 possible pulmonary embolism, 1 injection site hematoma, and 1 hospitalization unrelated to the DVT. CONCLUSION: Diagnosis of proximal DVT is straightforward, but calf DVT often requires venographic confirmation because of discrepancies among clinical probability, D-dimer estimation, and ultrasound scan. Outpatient treatment can be offered to patients who can comply with the regimen. The quality of anticoagulation is in accordance with published data, and compliance with external leg compression is almost perfect.