Thyroid status affects rat liver regeneration after partial hepatectomy by regulating cell cycle and apoptosis proteins

In rats, various growth factors and hormones, as well as partial hepatectomy (PH) are able to trigger the proliferative response of hepatocytes. Although recent evidence highlights the important role of thyroid hormones and thyroid status in regulating the growth of liver cells in vitro and in vivo models, the mechanism involved in the pro-proliferative effects of thyroid hormones is still unclear. Here we have investigated how in rats made hypo- and hyperthyroid after prolonged treatment respectively with propylthiouracil (PTU) and triiodothyronine (T3), the thyroid status affects liver regeneration after PH by regulating cell cycle and apoptosis proteins. Our results show that both in control and partially hepatectomized animals hyperthyroidism increases the cyclin D1, E and A levels and the activity of cyclin-cdk complexes, and decreases the levels of cdk inhibitors such as p16 and p27. On the contrary hypothyroidism induces a down-regulation of the activity of cyclin cdk complexes decreasing cyclin levels. Thyroid hormones control also p53 and p73, two proteins involved in apoptosis and growth arrest which are induced by PH. In particular, hypothyroidism increases and T3 treatment decreases p73 levels. The analysis of the phosphorylated forms of p42/44 and p38 MAPK revealed that they are induced during hepatic regeneration in euthyroid and hyperthyroid rats whereas they are negatively regulated in hypothyroid rats. In conclusion our data demonstrate that thyroid status can affects liver regeneration, altering the expression and the activity of the proteins involved in the control of cell cycle and growth arrest. Copyright © 2005 S. Karger AG, Basel

Murine obscurin and Obsl1 have functionally redundant roles in sarcolemmal integrity, sarcoplasmic reticulum organization, and muscle metabolism.

Biological roles of obscurin and its close homolog Obsl1 (obscurin-like 1) have been enigmatic. While obscurin is highly expressed in striated muscles, Obsl1 is found ubiquitously. Accordingly, obscurin mutations have been linked to myopathies, whereas mutations in Obsl1 result in 3M-growth syndrome. To further study unique and redundant functions of these closely related proteins, we generated and characterized Obsl1 knockouts. Global Obsl1 knockouts are embryonically lethal. In contrast, skeletal muscle-specific Obsl1 knockouts show a benign phenotype similar to obscurin knockouts. Only deletion of both proteins and removal of their functional redundancy revealed their roles for sarcolemmal stability and sarcoplasmic reticulum organization. To gain unbiased insights into changes to the muscle proteome, we analyzed tibialis anterior and soleus muscles by mass spectrometry, uncovering additional changes to the muscle metabolism. Our analyses suggest that all obscurin protein family members play functions for muscle membrane systems

Calcium Homeostasis Is Modified in Skeletal Muscle Fibers of Small Ankyrin1 Knockout Mice

Small Ankyrins (sAnk1) are muscle-specific isoforms generated by the Ank1 gene that participate in the organization of the sarcoplasmic reticulum (SR) of striated muscles. Accordingly, the volume of SR tubules localized around the myofibrils is strongly reduced in skeletal muscle fibers of 4- and 10-month-old sAnk1 knockout (KO) mice, while additional structural alterations only develop with aging. To verify whether the lack of sAnk1 also alters intracellular Ca2+ handling, cytosolic Ca2+ levels were analyzed in stimulated skeletal muscle fibers from 4- and 10-month-old sAnk1 KO mice. The SR Ca2+ content was reduced in sAnk1 KO mice regardless of age. The amplitude of the Ca2+ transients induced by depolarizing pulses was decreased in myofibers of sAnk1 KO with respect to wild type (WT) fibers, while their voltage dependence was not affected. Furthermore, analysis of spontaneous Ca2+ release events (sparks) on saponin-permeabilized muscle fibers indicated that the frequency of sparks was significantly lower in fibers from 4-month-old KO mice compared to WT. Furthermore, both the amplitude and spatial spread of sparks were significantly smaller in muscle fibers from both 4- and 10-month-old KO mice compared to WT. These data suggest that the absence of sAnk1 results in an impairment of SR Ca2+ release, likely as a consequence of a decreased Ca2+ store due to the reduction of the SR volume in sAnk1 KO muscle fibers

Functional Electrical Stimulation: A Possible Strategy to Improve Muscle Function in Central Core Disease?

Central Core Disease (CCD) is a congenital myopathy characterized by presence of amorphous central areas (or cores) lacking glycolytic/oxidative enzymes and mitochondria in skeletal muscle fibers. Most CCD families are linked to mutations in ryanodine receptor type-1 (RYR1), the gene encoding for the sarcoplasmic reticulum (SR) Ca2+ release channel of skeletal muscle. As no treatments are available for CCD, currently management of patients is essentially based on a physiotherapic approaches. Functional electrical stimulation (FES) is a technique used to deliver low energy electrical impulses to artificially stimulate selected skeletal muscle groups. Here we tested the efficacy of FES in counteracting muscle loss and improve function in the lower extremities of a 55-year-old female patient which was diagnosed with CCD at the age of 44. Genetic screening of the RyR1 gene identified a missense mutation (c.7354C>T) in exon 46 resulting in an amino acid substitution (p.R2452W) and a duplication (c.12853_12864dup12) in exon 91. The patient was treated with FES for 26 months and subjected before, during, and after training to a series of functional and structural assessments: measurement of maximum isometric force of leg extensor muscles, magnetic resonance imaging, a complete set of functional tests to assess mobility in activities of daily living, and analysis of muscle biopsies by histology and electron microscopy. All results point to an improvement in muscle structure and function induced by FES suggesting that this approach could be considered as an additional supportive measure to maintain/improve muscle function (and possibly reduce muscle loss) in CCD patients

Isolamento e caratterizzazione di sottopopolazioni ES-simili dal sangue di cordone ombelicale e periferico umani e dal midollo osseo murino

During the last few years much progress has been made in the knowledge of adult and embryonic stem cell biological properties. The ability to isolate and culture in conditions that allow the expansion of these cells without their losing stem potential, has helped in using stem cells as a tool to treat patients suffering from certain cellular degenerative diseases or affected with a specific cellular population loss of functionality. Because of their wide potentialities, embryonic stem cells (ES) obtained from blastocyst inner cell mass, appear to have the best chance as a tool to treat degenerative diseases affecting tissues of different embryonic origin. Biological difficulties (such as the risk of uncontrolled proliferation, graft versus host disease, the need to set culture conditions free of non-human compounds) and, above all, legal and ethical problems, seem to prevent the therapeutic application of such cells. The identification and characterization of an ES-like cell population in adult tissues might be of great help to exploit these cells in the medical field. In this thesis I show that a cellular population featuring certain molecular ES cell properties, may be isolated from human cord blood after depletion of all differentiated cells but monocytes, followed by the positive selection of cells bearing p75 neurotrophin receptor (p75NTR). Cytofluorimetric assays revealed that a fraction of p75NTR+ cells express SSEA3 and SSEA4, two typical human ES cell glycolipidic markers. Real Time RT-PCR assays showed that, following the selection for p75NTR+, a cell population is obtained that expresses very high levels of Oct4 and Nanog, two transcription factors whose importance in ES stemness and self-renewal is largely documented. Further increase of Oct4 and Nanog expression level is obtained following depletion of cells bearing Mac-1, a typical monocytes marker. A similar study has been made in human peripheral blood and mouse bone marrow. Data obtained by Real Time RT-PCR assays, have shown that in two out of four samples of adult peripheral blood analysed by the above selection method, following isolation of p75NTR+ cells, a cell population expressing high levels of Oct4 and Nanog was obtained. In 4-5 weeks-old mouse bone marrow, the highest expression of Oct4 and Nanog has been detected in p75NTR+/Mac-1- cells, while, in older mice (13-14 weeks), such expression increase has been detected in p75NTR+/Mac-1+ cells. Further studies are needed to evaluate whether these cells, in addition to bearing ES cell molecular markers, also have functional ES-like properties and can be classified as true ES-like cell

Calcium Homeostasis Is Modified in Skeletal Muscle Fibers of Small Ankyrin1 Knockout Mice

Small Ankyrins (sAnk1) are muscle-specific isoforms generated by the Ank1 gene that participate in the organization of the sarcoplasmic reticulum (SR) of striated muscles. Accordingly, the volume of SR tubules localized around the myofibrils is strongly reduced in skeletal muscle fibers of 4- and 10-month-old sAnk1 knockout (KO) mice, while additional structural alterations only develop with aging. To verify whether the lack of sAnk1 also alters intracellular Ca2+ handling, cytosolic Ca2+ levels were analyzed in stimulated skeletal muscle fibers from 4- and 10-month-old sAnk1 KO mice. The SR Ca2+ content was reduced in sAnk1 KO mice regardless of age. The amplitude of the Ca2+ transients induced by depolarizing pulses was decreased in myofibers of sAnk1 KO with respect to wild type (WT) fibers, while their voltage dependence was not affected. Furthermore, analysis of spontaneous Ca2+ release events (sparks) on saponin-permeabilized muscle fibers indicated that the frequency of sparks was significantly lower in fibers from 4-month-old KO mice compared to WT. Furthermore, both the amplitude and spatial spread of sparks were significantly smaller in muscle fibers from both 4- and 10-month-old KO mice compared to WT. These data suggest that the absence of sAnk1 results in an impairment of SR Ca2+ release, likely as a consequence of a decreased Ca2+ store due to the reduction of the SR volume in sAnk1 KO muscle fibers

Not all pericytes are born equal: Pericytes from human adult tissues present different differentiation properties

Pericytes (PCs) have been recognized for a long time only as structural cells of the blood vessels. The identification of tight contacts with endothelial cells and the ability to interact with surrounding cells through paracrine signaling revealed additional functions of PCs in maintaining the homeostasis of the perivascular environment. PCs got the front page, in the late 1990s, after the identification and characterization of a new embryonic cell population, the mesoangioblasts, from which PCs present in the adult organism are thought to derive. From these studies, it was clear that PCs were also endowed with multipotent mesodermal abilities. Furthermore, their ability to cross the vascular wall and to reconstitute skeletal muscle tissue after systemic injection opened the way to a number of studies aimed to develop therapeutic protocols for a cell therapy of muscular dystrophy. This has resulted in a major effort to characterize pericytic cell populations from skeletal muscle and other adult tissues. Additional studies also addressed their relationship with other cells of the perivascular compartment and with mesenchymal stem cells. These data have provided initial evidence that PCs from different adult tissues might be endowed with distinctive differentiation abilities. This would suggest that the multipotent mesenchymal ability of PCs might be restrained within different tissues, likely depending on the specific cell renewal and repair requirements of each tissue. This review presents current knowledge on human PCs and highlights recent data on the differentiation properties of PCs isolated from different adult tissues

Mesenchymal stem cells: from the perivascular environment to clinical applications

Adult stem cells represent a fundamental biological system that has fascinated scientists over the last decades, and are currently the subject of a large number of studies aimed at better defining the properties of these cells, with a prominent focus on improving their application in regenerative medicine. One of the most used adult stem cells in clinical trials are mesenchymal stem cells (MSCs), which are multipotent cells able to differentiate into mature cells of mesodermal lineages. Following the initial studies on MSCs isolated from bone marrow, similar cells were also isolated from a variety of fetal and adult human tissues. Initially considered as identical and equipotent, MSCs from tissues other than bone marrow actually display differences in terms of their plastic abilities, which can be ascribed to the tissue of origin and/or to the procedures used for their isolation. Moreover, results from additional studies suggest that cultured MSCs represent the in vitro version of a subset of in vivo resident cells localized in the perivascular environment. In this review, we will focus our attention on MSCs from tissues other than bone marrow, their in vivo localization and their current applications in clinics

Not all pericytes are born equal: Pericytes from human adult tissues present different differentiation properties

Pericytes (PCs) have been recognized for a long time only as structural cells of the blood vessels. The identification of tight contacts with endothelial cells and the ability to interact with surrounding cells through paracrine signaling revealed additional functions of PCs in maintaining the homeostasis of the perivascular environment. PCs got the front page, in the late 1990s, after the identification and characterization of a new embryonic cell population, the mesoangioblasts, from which PCs present in the adult organism are thought to derive. From these studies, it was clear that PCs were also endowed with multipotent mesodermal abilities. Furthermore, their ability to cross the vascular wall and to reconstitute skeletal muscle tissue after systemic injection opened the way to a number of studies aimed to develop therapeutic protocols for a cell therapy of muscular dystrophy. This has resulted in a major effort to characterize pericytic cell populations from skeletal muscle and other adult tissues. Additional studies also addressed their relationship with other cells of the perivascular compartment and with mesenchymal stem cells. These data have provided initial evidence that PCs from different adult tissues might be endowed with distinctive differentiation abilities. This would suggest that the multipotent mesenchymal ability of PCs might be restrained within different tissues, likely depending on the specific cell renewal and repair requirements of each tissue. This review presents current knowledge on human PCs and highlights recent data on the differentiation properties of PCs isolated from different adult tissues

The potential of obscurin as a therapeutic target in muscle disorders

Introduction: Obscurin, a giant protein of striated muscles, is emerging as an important player in a wide range of processes including myofibril assembly and maintenance, muscle protein degradation and intracellular signaling. Accordingly, obscurin participates to the mechanisms by which muscles adapt to physiological requirements or to pathological cues associated with cardiac and skeletal muscle diseases. Areas covered: The structure of the different obscurin isoforms identified so far, their tissue distribution and the most recent findings on obscurin in invertebrates and mammals will be reviewed. We will provide a synopsis of known molecular interactions between obscurin and other proteins and the biological relevance of these interactions for striated muscle function. The involvement of obscurin in protein degradation mechanisms and intracellular signaling will be also discussed along with initial evidence of a role of obscurin in the pathophysiology of human diseases. Expert opinion: Although still much remains to be discovered about the role of obscurin either as a structural component of the sarcomere or as a mediator of signaling pathways within muscle cells, it can be envisioned that this protein represents an interesting novel pharmacological target for the prevention and treatment of cardiac and skeletal muscle diseases