Cristina Giacinti

Local expression of IGF-1 accelerates muscle regeneration by rapidly modulating inflammatory cytokines and chemokines

Muscle regeneration following injury is characterized by myonecrosis accompanied by local inflammation, activation of satellite cells, and repair of injured fibers. The resolution of the inflammatory response is necessary to proceed toward muscle repair, since persistence of inflammation often renders the damaged muscle incapable of sustaining efficient muscle regeneration. Here, we show that local expression of a muscle‐restricted insulin‐like growth factor (IGF)‐1 (mIGF‐1) transgene accelerates the regenerative process of injured skeletal muscle, modulating the inflammatory response, and limiting fibrosis. At the molecular level, mIGF‐1 expression significantly down‐regulated proinflammatory cytokines, such as tumor necrosis factor (TNF)‐alpha and interleukin (IL)‐1beta, and modulated the expression of CC chemokines involved in the recruitment of monocytes/macrophages. Analysis of the underlying molecular mechanisms revealed that mIGF‐1 expression modulated key players of inflammatory response, such as macrophage migration inhibitory factor (MIF), high mobility group protein‐1 (HMGB1), and transcription NF‐KB. The rapid restoration of injured mIGF‐1 transgenic muscle was also associated with connective tissue remodeling and a rapid recovery of functional properties. By modulating the inflammatory response and reducing fibrosis, supplemental mIGF‐1 creates a qualitatively different environment for sustaining more efficient muscle regeneration and repair.—Pelosi, L., Giacinti, C., Nardis, C., Borsellino, G., Rizzuto, E., Nicoletti, C., Wannenes, F., Battistini, L., Rosenthal, N., Molinaro, M., Musaro, A. Local expression of IGF‐1 accelerates muscle regeneration by rapidly modulating inflammatory cytokines and chemokines. FASEB J. 21, 1393–1402 (2007)

Vascular Endothelial Growth Factor Modulates Skeletal Myoblast Function

Vascular endothelial growth factor (VEGF) expression is enhanced in ischemic skeletal muscle and is thought to play a key role in the angiogenic response to ischemia. However, it is still unknown whether, in addition to new blood vessel growth, VEGF modulates skeletal muscle cell function. In the present study immunohistochemical analysis showed that, in normoperfused mouse hindlimb, VEGF and its receptors Flk-1 and Flt-1 were expressed mostly in quiescent satellite cells. Unilateral hindlimb ischemia was induced by left femoral artery ligation. At day 3 and day 7 after the induction of ischemia, Flk-1 and Flt-1 were expressed in regenerating muscle fibers and VEGF expression by these fibers was markedly enhanced. Additional in vitro experiments showed that in growing medium both cultured satellite cells and myoblast cell line C2C12 expressed VEGF and its receptors. Under these conditions, Flk-1 receptor exhibited constitutive tyrosine phosphorylation that was increased by VEGF treatment. During myogenic differentiation Flk-1 and Flt-1 were down-regulated. In a modified Boyden Chamber assay, VEGF enhanced C2C12 myoblasts migration approximately fivefold. Moreover, VEGF administration to differentiating C2C12 myoblasts prevented apoptosis, while inhibition of VEGF signaling either with selective VEGF receptor inhibitors (SU1498 and CB676475) or a neutralizing Flk-1 antibody, enhanced cell death approximately 3.5fold. Finally, adenovirus-mediated VEGF165 gene transfer inhibited ischemia-induced apoptosis in skeletal muscle. These results support a role for VEGF in myoblast migration and survival, and suggest a novel autocrine role of VEGF in skeletal muscle repair during ischemia. (Am J Pathol 2003, 163:1417–1428)

p21WAF1 expression induced by MEK/ERK pathway activation or inhibition correlates with growth arrest, myogenic differentiation and onco-phenotype reversal in rhabdomyosarcoma cells

Backgroundp21WAF1, implicated in the cell cycle control of both normal and malignant cells, can be induced by p53-dependent and independent mechanisms. In some cells, MEKs/ERKs regulate p21WAF1 transcriptionally, while in others they also affect the post-transcriptional processes. In myogenic differentiation, p21WAF1 expression is also controlled by the myogenic transcription factor MyoD. We have previously demonstrated that the embryonal rhabdomyosarcoma cell line undergoes growth arrest and myogenic differentiation following treatments with TPA and the MEK inhibitor U0126, which respectively activate and inhibit the ERK pathway.In this paper we attempt to clarify the mechanism of ERK-mediated and ERK-independent growth arrest and myogenic differentiation of embryonal and alveolar rhabdomyosarcoma cell lines, particularly as regards the expression of the cell cycle inhibitor p21WAF1.Resultsp21WAF1 expression and growth arrest are induced in both embryonal (RD) and alveolar (RH30) rhabdomyosarcoma cell lines following TPA or MEK/ERK inhibitor (U0126) treatments, whereas myogenic differentiation is induced in RD cells alone. Furthermore, the TPA-mediated post-transcriptional mechanism of p21WAF1-enhanced expression in RD cells is due to activation of the MEK/ERK pathway, as shown by transfections with constitutively active MEK1 or MEK2, which induces p21WAF1 expression, and with ERK1 and ERK2 siRNA, which prevents p21WAF1 expression. By contrast, U0126-mediated p21WAF1 expression is controlled transcriptionally by the p38 pathway. Similarly, myogenin and MyoD expression is induced both by U0126 and TPA and is prevented by p38 inhibition. Although MyoD and myogenin depletion by siRNA prevents U0126-mediated p21WAF1 expression, the over-expression of these two transcription factors is insufficient to induce p21WAF1. These data suggest that the transcriptional mechanism of p21WAF1 expression in RD cells is rescued when MEK/ERK inhibition relieves the functions of myogenic transcription factors. Notably, the forced expression of p21WAF1 in RD cells causes growth arrest and the reversion of anchorage-independent growth.ConclusionOur data provide evidence of the key role played by the MEK/ERK pathway in the growth arrest of Rhabdomyosarcoma cells. The results of this study suggest that the targeting of MEK/ERKs to rescue p21WAF1 expression and myogenic transcription factor functions leads to the reversal of the Rhabdomyosarcoma phenotype.

Human cardiac progenitor cell grafts as unrestricted source of supernumerary cardiac cells in healthy murine hearts.

Human heart harbors a population of resident progenitor cells that can be isolated by stem cell antigen‐1 antibody and expanded in culture. These cells can differentiate into cardiomyocytes in vitro and contribute to cardiac regeneration in vivo. However, when directly injected as single cell suspension, less than 1%‐5% survive and differentiate. Among the major causes of this failure are the distressing protocols used to culture in vitro and implant progenitor cells into damaged hearts. Human cardiac progenitors obtained from the auricles of patients were cultured as scaffoldless engineered tissues fabricated using temperature‐responsive surfaces. In the engineered tissue, progenitor cells established proper three‐dimensional intercellular relationships and were embedded in self‐produced extracellular matrix preserving their phenotype and multipotency in the absence of significant apoptosis. After engineered tissues were leant on visceral pericardium, a number of cells migrated into the murine myocardium and in the vascular walls, where they integrated in the respective textures.

Scriptaid effects on breast cancer cell lines

In breast cancer tumor expression of estrogen receptors (ERs) is important as a marker of prognosis and mostly as a predictor of response to endocrine therapy. In fact, the loss of α‐ER expression leads to unresponsiveness to anti‐hormone treatment. In a significant fraction of breast cancers, this loss of expression is a result of epigenetic mechanisms, such as DNA methylation and histone deacetylation, within the α‐ER promoter. Previous studies have shown that pharmacologic inhibition of these mechanisms using the DNA methyltransferase inhibitor, 5‐aza‐2‐deoxycytidine (AZA), and the histone deacetylase (HDAC) inhibitor, Trichostatin A (TSA), results in expression of functional α‐ER mRNA and protein. Moreover, the activity of a novel HDAC inhibitor, Scriptaid, has been shown to induce inhibition of tumor growth in breast cancer and to cause re‐expression of functional α‐ER in α‐ER negative breast cancer cells. We sought to better characterize the effects of Scriptaid on cell growth, apoptosis, and α‐ER expression in α‐ER‐positive (MCF‐7), α‐ER‐negative (MDA‐MB‐231), and α‐ER‐negative/Her‐2 over‐expressing (SKBr‐3) human breast cancer cell lines. In all of these cell lines Scriptaid treatment resulted in significant growth inhibition and apoptosis, and RT‐PCR confirmed an increase of α‐ER mRNA transcript in MDA‐MB‐231 after 48 h of Scriptaid treatment. Furthermore, following treatment with Scriptaid, the formerly unresponsive MDA‐MB‐231 and SKBr‐3 breast cancer cells became responsive to tamoxifen. These results show that the HDAC inhibitor Scriptaid is able to sensitize tamoxifen hormone‐resistant breast cancer cells, and that Scriptaid or related HDAC inhibitors are candidates for further study in breast cancer. J. Cell. Physiol. 227: 3426–3433, 2012.

Stem cell-mediated muscle regeneration and repair in aging and neuromuscular diseases

One of the most exciting aspirations of current medical science is the regeneration of damaged body parts. The capacity of adult tissues to regenerate in response to injury stimuli represents an important homeostatic process that until recently was thought to be limited in mammals to tissues with high turnover such as blood and skin. However, it is now generally accepted that each tissue type, even those considered post-mitotic, such as nerve or muscle, contains a reserve of undifferentiated progenitor cells, loosely termed stem cells, participating in tissue regeneration and repair. Skeletal muscle regeneration is a coordinate process in which several factors are sequentially activated to maintain and preserve muscle structure and function upon injury stimuli. In this review, we will discuss the role of stem cells in muscle regeneration and repair and the critical role of specific factors, such as IGF-1, vasopressin and TNF-alpha, in the modulation of the myogenic program and in the regulation of muscle regeneration and homeostasis.Branching morphogenesis is a multi-step process that controls the formation of polarised tubules starting from hollow cysts. Its execution entails a series of rate-limiting events which include reversible disruption of cell polarity, dismantling of intercellular contacts, acquisition of a motile phenotype, stimulation of cell proliferation, and final re-establishment of cell polarity for creation of the definitive structures. Branching morphogenesis takes place physiologically during development, accounting for the establishment of organs endowed with a ramified architecture such as glands, the respiratory tract and the vasculartree. In cancer, aberrant implementation of branching morphogenesis leads to deregulated proliferation, protection from apoptosis and enhanced migratory/invasive properties, which together exacerbate the aggressive features of neoplastic cells. Under both physiological and pathological conditions, branching morphogenesis is mainly accomplished by a family of growth factors known as scatter factors. In this review, we will summarise the current knowledge on the biological and functional roles of scatter factors during branching morphogenesis, with a special emphasis on the phenotypic (structural and histological) consequences of scatter factor activity in different tissues.We present here findings obtained on a large number of human tissues over a period of more than ten years, by our modification of the Osmium maceration method for high resolution scanning electron microscopy (HRSEM). Data are documented by original pictures which illustrate both some 3-D intracellular features not previously shown in human tissues, and results obtained in our current studies on mitochondrial morphology and on the secretory process of salivary glands. We have demonstrated that mitochondria of cells of practically all human tissues and organs have usually tubular cristae, and that even the cristae that look lamellar are joined to the inner mitochondrial membrane by tubular connexions similar to the crista junctions later seen by electron tomography. Concerning salivary glands an important result is the development of a morphometric method that allows the quantitative evaluation of the secretory events.

Cdk9-55: A new player in muscle regeneration

Adult skeletal muscle contains a specialized population of myogenic quiescent stem cells, termed satellite cells, which contribute to repair myofibers after injury. During muscle regeneration, satellite cells exit their normal quiescent state, proliferate, activating MyoD and Myf‐5 expression, and finally differentiate and fuse to reconstitute the injured muscle architecture. We have previously reported that cdk9 is required for myogenesis in vitro by activating MyoD‐dependent transcription. In myoblasts induced to differentiate, MyoD recruits cdk9 on the chromatin of muscle‐specific regulatory regions. This event correlates with chromatin‐modifying enzyme recruitment and phosphorylation of cdk9‐specific target residues at the carboxyl‐terminal domain of RNA polymerase II. Here we report that a second cdk9 isoform, termed cdk9‐55, plays a fundamental role in muscle regeneration and differentiation in vivo. This alternative form is specifically induced in injured myofibers and its activity is strictly required for the completion of muscle regeneration process. J. Cell. Physiol. 216: 576–582, 2008,

Muscle Involvement and IGF-1 Signaling in Genetic Disorders: New Therapeutic Approaches

In the last decade, dramatic progress has been made in elucidating the molecular defects underlying a number of muscle diseases. With the characterization of mutations responsible for muscle dysfunction in several inherited pathologies, and the identification of novel signaling pathways, subtle alterations in which can lead to significant defects in muscle metabolism, the field is poised to devise successful strategies for treatment of this debilitating and often fatal group of human ailments. Yet progress has been slow in therapeutic applications of our newly gained knowledge. The complexity of muscle types, the intimate relationship between structural integrity and mechanical function, and the sensitivity of skeletal muscle to metabolic perturbations have impeded rapid progress in successful clinical intervention. The relatively poor regenerative properties of striated muscle compound the devastating effects of muscle degeneration. Perhaps the most difficult hurdle is the sheer volume of tissue that must be treated to effect a significant improvement in quality of life. Recent studies on the role of insulin-like growth factor-1 in skeletal muscle growth and homeostasis have excited new interest in this important mediator of anabolic pathways and suggest promising new avenues for intervention in catabolic disease. In this review, we will discuss the potential therapeutic role of local insulin-like growth factor 1 in the treatment of muscle wasting associated with muscle diseases.

Cell Cycle Regulation in Myogenesis

Skeletal myoblasts differentiation begins with irreversible withdrawal of myoblasts from the cell cycle. This growth arrest is mediated and maintained by the Rb protein, together with p21 and other inhibitors of cell cycle progression. The subsequent activation of muscle-specific promoters at the onset of differentiation is regulated by myogenic bHLH transcription factors, such as MyoD. MyoD protein binds DNA and recruits coactivators and corepressors of transcription to specific promoters and orchestrates the early differentiation events that lead to the fusion of myoblasts into myotubes and the formation of skeletal muscle.