E. Lesma - Academia.edu (original) (raw)

Papers by E. Lesma

Research paper thumbnail of Pharmacological reversion of the phenotype of human TSC2-/- smooth muscle cells

Research paper thumbnail of Matrix methalloproteinase 2 and 7 are new targets in LAM and TSC-related disorders

Tuberous sclerosis complex (TSC), an autosomal dominant disease characterized by the formation of... more Tuberous sclerosis complex (TSC), an autosomal dominant disease characterized by the formation of hamartomas in various organs, is caused by mutations in TSC1 e TSC2 tumor suppressor genes, encoding hamartin and tuberin, respectively. Lymphangioleiomyomatosis (LAM) is a rare lung disease characterized by cystic lung destruction and resulting from proliferation of neoplastic cells bearing mutations in either TSC1 or TSC2 genes. A metastatic process has been proposed in dissemination of LAM and TSC cells to explain the ability to cause hamartomas in various organs. Matrix metalloproteinases (MMPs) degrade and modify the extracellular matrix (ECM), facilitating detachment of cells from the tissue. Levels of MMP-2 and MMP-9 in urine have been proposed to be predictive of disease status in a variety of cancers and also in LAM. MMP-2 and MMP-9 levels are high in urine of LAM patients and MMP-2 is substantially up-regulated in their lung tissue (Glasgow et al.,2010). Matrilisin (MMP-7) con...

Research paper thumbnail of Molecular characterization of growth and survival of human TSC2 gene-deficient smooth muscle-like cells

Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome resulting from the loss of f... more Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome resulting from the loss of function of the TSC1/2 complex. The TSC1 and TSC2 gene products, hamartin and tuberin, associate as a functional complex and inhibit the mammalian target of rapamycin (mTOR). The loss of tuberin function leads to constitutive S6K1 activation and phosphorylation of its ribosomal substrate S6. We have recently reported the isolation from a renal angiomyolipoma and characterization of smooth muscle-like cells (A+) showing loss of heterozigosity for the TSC2 gene. A+ cells require epidermal growth factor (EGF) supplementation to sustain proliferation, and exposure to antibodies to EGF receptor (EGFR) is lethal. In addition, A+ cells secrete insulin-like growth factor-1 (IGF-1). Incubation at plating time with rapamycin (1 and 5ng/ml) for 24 and 48 hours decreases phosphorylation of S6 in a dose- and time-dependent manner and normalizes A+ cell proliferation. Similar results were obtained afte...

Research paper thumbnail of TSC2 smooth muscle-like cells from human angiomyolipoma require EGF for growth while rapamycin is inactive

Research paper thumbnail of Characterization of smooth muscle-like cells derived from TSC2 human renal angiomyolipoma and their phenotype reversion

Global LAM Summit, Feb 25, 2007

Research paper thumbnail of Brain tubers in TSC2 patients express the anti-apoptotic protein survivin

Meeting of the Society for Neuroscience, Nov 12, 2005

Research paper thumbnail of Gruppo di studio multidisciplinare per la TSC : Risultati preliminari = Multicentric study group for tuberous sclerosis complex : Preliminary results

Research paper thumbnail of Adipose-Derived Stem Cells from Fat Tissue of Breast Cancer Microenvironment Present Altered Adipogenic Differentiation Capabilities

Stem Cells International, 2019

Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate into multiple cell type... more Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate into multiple cell types, including adipocytes, osteoblasts, and chondrocytes. The role of adipose-derived stem cells (ADSCs) in cancers is significantly relevant. They seem to be involved in the promotion of tumour development and progression and relapse processes. For this reason, investigating the effects of breast cancer microenvironment on ADSCs is of high importance in order to understand the relationship between tumour cells and the surrounding stromal cells. With the current study, we aimed to investigate the specific characteristics of human ADSCs isolated from the adipose tissue of breast tumour patients. We compared ADSCs obtained from periumbilical fat (PF) of controls with ADSCs obtained from adipose tissue of breast cancer- (BC-) bearing patients. We analysed the surface antigens and the adipogenic differentiation ability of both ADSC populations. C/EBPδ expression was increased in PF and BC ADSC...

Research paper thumbnail of Amelioration of Neutrophil Membrane Function Underlies Granulocyte-Colony Stimulating Factor Action in Glycogen Storage Disease 1b

International Journal of Immunopathology and Pharmacology, 2005

Glycogen storage disease (GSD) 1b is a metabolic disorder characterized by a deficiency of glucos... more Glycogen storage disease (GSD) 1b is a metabolic disorder characterized by a deficiency of glucose 6-phosphate transporter and neutrophil alterations, which are reduced in number and functionally impaired. The present study aimed at investigating neutrophil dysfunction correlating submembrane and cytoskeletal changes at different ages with or without granulocyte-colony stimulating factor (G-CSF) treatment. GSD1b neutrophils showed reduced expression and diffused localization of focal adhesion kinase (FAK) and actin. No abnormalities were observed in GSD1a patient neutrophils. Gelsolin was also slightly reduced in neutrophils of GSD1b patients. When patients were treated for at least 3 months with G-CSF, the neutrophil number and the expression of FAK and actin were significantly increased. Granulocyte colony-stimulating factor treatment was similarly effective when performed in 1 year old patients. FAK auto- and IL-8-mediated phosphorylations were already affected as early as 1 year...

Research paper thumbnail of Promoter methylation of TSC2 gene is important for pathogenesis of TSC and LAM lesions

Research paper thumbnail of Pathogenesis and pharmacological evaluation of human TSC2 smooth muscle cells. Possible treatment options for TSC and lymphangioleiomyomatosis

Tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM) are rare genetic diseases cau... more Tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM) are rare genetic diseases caused by mutations in TSC genes. TSC2-/- cells form hamartomas and invade lungs causing the fatal disease LAM. We have isolated from an angiomyolipoma a homogenous population of TSC2 smooth muscle-like (ASM) cells with LOH genetic lesion (TSC2-/-ASM cells). These cells grow and proliferate in EGF-dependent manner, while auto-secreted IGF-1 acts as a surviving factor. The constitutive phosphorylation of S6, and higher Akt and ERK phosphorylation are a paradigm of these cells. We validated our cells as a cellular model for studying TSC and LAM by TSC2 gene transfection, which promoted the biochemical normalization and eliminated EGF-dependency. The blockade of EGF-receptors with specific antibodies results in TSC2-/- ASM cells death and suggest a novel therapeutic avenue for TSC and LAM. We have recently found that suppression of TSC2 gene may also have an epigenetic origin, another pure ASM cell population was isolated in our lab. These cells showed the typical constitutive S6 phosphorylation, and lacked tuberin as the result of TSC2 promoter methylation as a second hit (TSC2-/methASM cells). Chromatin remodelling agents, such as trichostatin-A and 5-azacytidine de-methylated TSC2-/methASM cell promoter, that returned normal. TSC2-/methASM cells are pharmacologically comparable to TSC2-/-ASM cells. These data show for the first time that TSC2 pathogenesis might be originated also by epigenetic defects in ASM cells. Pulmonary proliferation of TSC2-/-ASM cells in LAM is characterized by cystic destruction of lung parenchyma. To develop a proper animal model for LAM and to validate our in vitro pharmacological observations we have administered by endonasal route TSC2-/-ASM cells to nude mice. TSC2-/-ASM cells massively penetrated into lungs, and from there reached the lymphatic vessels where they accumulated in the lymph nodes. Here these cells grow and proliferate. The progressive increase of TSC2-/-ASM cells in lung parenchyma correspond to the progressive invasion of lymphatic vessel and destruction of lung tissue. Anti-EGFR antibody treatment killed TSC2-/-ASM cells and reversed lung structural alterations, while rapamycin had a weaker effect. These data suggest a novel therapeutic approach for the control of the abnormal ASM cell growth in TSC and lymphangioleiomyomatosi

Research paper thumbnail of Motility and Invasive Properties of Lam/TSC Cells from Chylous of a Lam/TSC Patient

Mutations in TSC2 are common factors in the development of TSC. LAM can occur as a sporadic disor... more Mutations in TSC2 are common factors in the development of TSC. LAM can occur as a sporadic disorder or associated with TSC. We have isolated human TSC2-smooth-muscle-cells from chylous of a LAM/TSC patient (LAM/TSC cells). In these cells tuberin expression is related to a TSC2 epigenetic regulation, and proliferation and survival are EGF-dependent. Cancer metastasis results in altered cell motility and invasiveness and is modulated by Notch activity. Given the fact that LAM/TSC cells express mesenchymal features, and survive in adherent or nonadherent status, we studied their invasive properties and motility. LAM/TSC cells showed anoikis resistence in soft agar assay confirming their loss of contact inhibition and acquisition of anchorage independent survival. Cell motility appear to be related to tuberin expression since it was reduced by 5-azacytidine treatment. Rapamycin and anti-EGFR decreased the migration rate but at a slightly lower extent than 5-azacytidine. Adherent and nonadherent LAM/TSC cells invaded epithelial cell layers by feeder cell invasion assay with an increased of IL-6 and IL-8 secretion. LAM/TSC cells express Notch and its targets (e.i. Hes, Jagged 1,2) that are not regulated by specific inhibitors. In conclusion, LAM/TSC cells express invasive and migratory features which appear to be related to tuberin expression

Research paper thumbnail of Anti-EGFR Antibody Reduces Lung Nodules by Inhibition of EGFR-Pathway in a Model of Lymphangioleiomyomatosis

BioMed Research International, 2015

EGFR belongs to the HER/ErbB family of tyrosine kinase receptors and its activation in cancer cel... more EGFR belongs to the HER/ErbB family of tyrosine kinase receptors and its activation in cancer cells has been linked with increased proliferation, angiogenesis, and metastasis. Lymphangioleiomyomatosis (LAM) is a rare, low-grade neoplasm that occurs sporadically or in association with tuberous sclerosis complex (TSC), a genetic, multisystem disorder characterized by hamartomas in several organs. From chylous of a LAM/TSC patient, we previously isolated smooth muscle-like LAM/TSC cells whose proliferation depends on EGF and monoclonal anti-EGFR antibodies reduced proliferation and caused cell death. We demonstrated that the dependency from EGF was caused by the absence of tuberin. To study the role of EGFR pathwayin vivo, we developed a mouse model by administration of LAM/TSC cells to female nude mice. LAM/TSC cells caused pulmonary airspace enlargement and, after 30 weeks, nodule formation which express EGFR. Anti-EGFR antibody decreased the number and dimension of lung nodules like...

Research paper thumbnail of Experimental diabetic neuropathy, inhibition of excessive protein mono-ADP-ribosylation prevents reduction of substance P axonal transport

Pharmacological Research, 1995

ABSTRACT

Research paper thumbnail of Chromatin Remodeling by Rosuvastatin Normalizes TSC2-/meth Cell Phenotype through the Expression of Tuberin

Journal of Pharmacology and Experimental Therapeutics, 2013

Tuberous sclerosis complex (TSC) is a multi-systemic syndrome caused by mutations in TSC1 or TSC2... more Tuberous sclerosis complex (TSC) is a multi-systemic syndrome caused by mutations in TSC1 or TSC2 gene. In TSC2null cells, Rheb, a member of the Ras family of GTPases, is constitutively activated. Statins inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase and block the synthesis of isoprenoid lipids with inhibition of Rheb farnesylation and RhoA geranylgeranylation. The effects of rosuvastatin on the function of human TSC2 2/2 and TSC2-/meth a-actin smooth muscle (ASM) cells have been investigated. The TSC2 2/2 and TSC2-/meth ASM cells, previously isolated in our laboratory from the renal angiomyolipoma of two TSC patients, do not express tuberin and bear loss of heterozigosity caused by a double hit on TSC2 and methylation of TSC2 promoter, respectively. Exposure to rosuvastatin affected TSC2-/meth ASM cell growth and promoted tuberin expression by acting as a demethylating agent. This occurred without changes in interleukin release. Rosuvastatin also reduced RhoA activation in TSC2-/meth ASM cells, and it required coadministration with the specific mTOR (mammalian target of rapamycin) inhibitor rapamycin to be effective in TSC2 2/2 ASM cells. Rapamycin enhanced rosuvastatin effect in inhibiting cell proliferation in TSC2 2/2 and TSC2-/meth ASM cells. Rosuvastatin alone did not alter phosphorylation of S6 and extracellular signal-regulated kinase (ERK), and at the higher concentration, rosuvastatin and rapamycin slightly decreased ERK phosphorylation. These results suggest that rosuvastatin may potentially represent a treatment adjunct to the therapy with mTOR inhibitors now in clinical development for TSC. In particular, rosuvastatin appears useful when the disease is originated by epigenetic defects.

Research paper thumbnail of Diabetic neuropathy in the rat: 1. Alcar augments the reduced levels and axoplasmic transport of substance P

Journal of Neuroscience Research, 1995

This study examined the sciatic nerve axonal transport of substance P‐like immunoreactivity (SPLI... more This study examined the sciatic nerve axonal transport of substance P‐like immunoreactivity (SPLI) and its basal content in stomach, sciatic nerve and lumbar spinal cord of 8‐ and 12‐week alloxan‐diabetic rats, respectively. One group of diabetic rats received acetyl‐l‐carnitine (ALCAR) throughout the experimental period. Alloxan treatment caused hyperglycemia and reduced body growth. Axonal transport of SPLI was studied by measurement of 24‐hour accumulation at a ligature on the sciatic nerve. There was a marked reduction (from 50% to 100% according to the nerve segment examined) of anterograde and retrograde accumulation of SPLI in the constricted nerve of 8‐week diabetic rats. In the sciatic nerve of ALCAR‐treated diabetic rats, the accumulation of SPLI was comparable to control values. In the sciatic nerve, lumbar spinal cord and stomach of 12‐week diabetic rats, there is a significant reduction of SPLI content. ALCAR treatment prevented SPLI loss in these tissues. Sciatic nerve...

Research paper thumbnail of Perinatal morphine II: Changes in cortical plasticity

Journal of Neuroscience Research, 1995

We have previously shown that perinatal exposure to morphine impairs reactive plasticity of serot... more We have previously shown that perinatal exposure to morphine impairs reactive plasticity of serotonin (5‐HT) neurons following selective neonatal lesion (Gorio et al., J Neurosci Res 34:462–471, 1993). This study shows that morphine inhibits also that the compensatory sprouting of intact axons after partial denervation. Neonatal 6‐OHDA injection causes norepinephrine (NE) depletion in the frontal cortex, which triggers a compensatory increase of dopamine, serotonin (5‐HT), and met‐enkephalin content correlated by the increased density of tyrosine hydroxylase– and 5‐HT–positive axons. In perinatal morphine‐treated rats, no compensatory changes are observed after neonatal 6‐OHDA depletion of NE in the frontal cortex. ©1995 Wiley‐Liss, Inc.

Research paper thumbnail of Inhibition of high glucose-induced protein mono-ADP-ribosylation restores neuritogenesis and sodium-pump activity in SY5Y neuroblastoma cells

Journal of Neuroscience Research, 1999

The exposure of SY5Y neuroblastoma cells to high concentrations of glucose, fructose, or galactos... more The exposure of SY5Y neuroblastoma cells to high concentrations of glucose, fructose, or galactose is an experimental model commonly used for in vitro evaluation of typical neuronal alterations observed in diabetes mellitus. In the present study, we observed that 2 weeks of exposure to high carbohydrate concentrations caused both a significant impairment in neurite formation induced by supplementation of retinoic acid or by subtraction of fetal calf serum to the culture medium and a marked reduction in Na(+)-K(+)-ATPase activity. However, only the exposure to high millimoles of glucose caused an enhancement of mono-ADP-ribosylation, typical of diabetes mellitus, affecting at least five proteins. The concomitant exposure to high glucose and to silybin, a mono-ADP-ribosylation inhibitor, normalized the extent of ADP-ribosylation of the five proteins and counteracted the inhibitory effects of high glucose on Na(+)-pump activity and on neuritogenesis. Conversely, the supplementation of silybin did not prevent fructose and galactose inhibitory effects on Na(+)-pump activity and neurite formation. These data confirm those of previous reports suggesting a link between excessive protein mono-ADP-ribosylation and the onset of diabetic complications such as diabetic neuropathy.

Research paper thumbnail of Glycosaminoglycans treatment increases IGF-I muscle levels and counteracts motor neuron death: A novel nonanticoagulant action

Journal of Neuroscience Research, 1999

The present study shows that sciatic nerve crush in 2-day-old rats causes extensor digitorum long... more The present study shows that sciatic nerve crush in 2-day-old rats causes extensor digitorum longus (EDL) muscle atrophy and motor neuron loss and that treatment with glycosaminoglycans (GAGs) promotes muscle reinnervation, motor neuron survival, and markedly increases insulin-like growth factor-I (IGF-I) content in the denervated muscles. EDL muscle denervation-induced atrophy in saline-treated rats is progressive and reaches the greatest extent at 42 days after birth, which correlates with reduced EDL weight growth. There is also a partial reinnervation as shown by the number of reinnervated EDL muscle fibers (65.4% of control) and by the poor restoration of the indirect isometric twitch tension (62% of control) that is further reduced under tetanic stimulation (34% of control). The number of surviving motor neurons that innervate EDL muscle drops from 55 +/- 3 to 29 +/- 8. In GAGs-treated 42-day-old rats, the effects of neonatal nerve lesioning on EDL muscle atrophy and denervation are successfully reversed, and the isometric twitch tension and the capacity to hold tetanic stimulation are restored to almost control levels. The number of surviving EDL motor neurons is also increased to 43 +/- 4. Treatment with GAGs selectively affects IGF-I content in denervated hindlimb muscles, which is augmented from 7.02 +/- 0.71 ng/mg tissue to 25.72 +/- 0.7 in the EDL and from 3.2 +/- 0.18 to a robust 211 +/- 9.6 in the soleus.

Research paper thumbnail of G protein-mediated signal transduction is affected in primary biliary cirrhosis

Hepatology Research, 2006

Primary biliary cirrhosis (PBC) is a chronic liver disease characterized by severe intraepatic ch... more Primary biliary cirrhosis (PBC) is a chronic liver disease characterized by severe intraepatic cholestasis. Pruritus often occurs during the course of the illness. We designed a study aimed at assessing whether pruritus is associated with dysfunction of signal transduction. Seventeen female patients affected by PBC were enrolled into the study and divided in two groups according to severity of liver disease. Leukocytes were isolated from peripheral blood and Gi, Go and Gs protein expressions were evaluated by western blotting, while G protein function was assessed by measuring cyclic adenosine phosphate formation. The expression of all types of G proteins was increased in leukocytes of PBC patients. The basal adenylate activity was significally higher than control in patients with less severe liver disease, while it was lower than normal in those with severe liver disease. Incubation of patient leukocytes with guanosine triphosphate-gamma-S and Gs protein activators failed to enhance cAMP production, while N-formyl-met-leu-phe was more effective in reducing cAMP production. The expression of all G proteins was non-selectively increased in PBC leukocytes, while adenylate cyclase activity was significantly modified. However, the observed changes in G proteins expression and in adenylate cyclase activity are not related to the presence of pruritus.

Research paper thumbnail of Pharmacological reversion of the phenotype of human TSC2-/- smooth muscle cells

Research paper thumbnail of Matrix methalloproteinase 2 and 7 are new targets in LAM and TSC-related disorders

Tuberous sclerosis complex (TSC), an autosomal dominant disease characterized by the formation of... more Tuberous sclerosis complex (TSC), an autosomal dominant disease characterized by the formation of hamartomas in various organs, is caused by mutations in TSC1 e TSC2 tumor suppressor genes, encoding hamartin and tuberin, respectively. Lymphangioleiomyomatosis (LAM) is a rare lung disease characterized by cystic lung destruction and resulting from proliferation of neoplastic cells bearing mutations in either TSC1 or TSC2 genes. A metastatic process has been proposed in dissemination of LAM and TSC cells to explain the ability to cause hamartomas in various organs. Matrix metalloproteinases (MMPs) degrade and modify the extracellular matrix (ECM), facilitating detachment of cells from the tissue. Levels of MMP-2 and MMP-9 in urine have been proposed to be predictive of disease status in a variety of cancers and also in LAM. MMP-2 and MMP-9 levels are high in urine of LAM patients and MMP-2 is substantially up-regulated in their lung tissue (Glasgow et al.,2010). Matrilisin (MMP-7) con...

Research paper thumbnail of Molecular characterization of growth and survival of human TSC2 gene-deficient smooth muscle-like cells

Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome resulting from the loss of f... more Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome resulting from the loss of function of the TSC1/2 complex. The TSC1 and TSC2 gene products, hamartin and tuberin, associate as a functional complex and inhibit the mammalian target of rapamycin (mTOR). The loss of tuberin function leads to constitutive S6K1 activation and phosphorylation of its ribosomal substrate S6. We have recently reported the isolation from a renal angiomyolipoma and characterization of smooth muscle-like cells (A+) showing loss of heterozigosity for the TSC2 gene. A+ cells require epidermal growth factor (EGF) supplementation to sustain proliferation, and exposure to antibodies to EGF receptor (EGFR) is lethal. In addition, A+ cells secrete insulin-like growth factor-1 (IGF-1). Incubation at plating time with rapamycin (1 and 5ng/ml) for 24 and 48 hours decreases phosphorylation of S6 in a dose- and time-dependent manner and normalizes A+ cell proliferation. Similar results were obtained afte...

Research paper thumbnail of TSC2 smooth muscle-like cells from human angiomyolipoma require EGF for growth while rapamycin is inactive

Research paper thumbnail of Characterization of smooth muscle-like cells derived from TSC2 human renal angiomyolipoma and their phenotype reversion

Global LAM Summit, Feb 25, 2007

Research paper thumbnail of Brain tubers in TSC2 patients express the anti-apoptotic protein survivin

Meeting of the Society for Neuroscience, Nov 12, 2005

Research paper thumbnail of Gruppo di studio multidisciplinare per la TSC : Risultati preliminari = Multicentric study group for tuberous sclerosis complex : Preliminary results

Research paper thumbnail of Adipose-Derived Stem Cells from Fat Tissue of Breast Cancer Microenvironment Present Altered Adipogenic Differentiation Capabilities

Stem Cells International, 2019

Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate into multiple cell type... more Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate into multiple cell types, including adipocytes, osteoblasts, and chondrocytes. The role of adipose-derived stem cells (ADSCs) in cancers is significantly relevant. They seem to be involved in the promotion of tumour development and progression and relapse processes. For this reason, investigating the effects of breast cancer microenvironment on ADSCs is of high importance in order to understand the relationship between tumour cells and the surrounding stromal cells. With the current study, we aimed to investigate the specific characteristics of human ADSCs isolated from the adipose tissue of breast tumour patients. We compared ADSCs obtained from periumbilical fat (PF) of controls with ADSCs obtained from adipose tissue of breast cancer- (BC-) bearing patients. We analysed the surface antigens and the adipogenic differentiation ability of both ADSC populations. C/EBPδ expression was increased in PF and BC ADSC...

Research paper thumbnail of Amelioration of Neutrophil Membrane Function Underlies Granulocyte-Colony Stimulating Factor Action in Glycogen Storage Disease 1b

International Journal of Immunopathology and Pharmacology, 2005

Glycogen storage disease (GSD) 1b is a metabolic disorder characterized by a deficiency of glucos... more Glycogen storage disease (GSD) 1b is a metabolic disorder characterized by a deficiency of glucose 6-phosphate transporter and neutrophil alterations, which are reduced in number and functionally impaired. The present study aimed at investigating neutrophil dysfunction correlating submembrane and cytoskeletal changes at different ages with or without granulocyte-colony stimulating factor (G-CSF) treatment. GSD1b neutrophils showed reduced expression and diffused localization of focal adhesion kinase (FAK) and actin. No abnormalities were observed in GSD1a patient neutrophils. Gelsolin was also slightly reduced in neutrophils of GSD1b patients. When patients were treated for at least 3 months with G-CSF, the neutrophil number and the expression of FAK and actin were significantly increased. Granulocyte colony-stimulating factor treatment was similarly effective when performed in 1 year old patients. FAK auto- and IL-8-mediated phosphorylations were already affected as early as 1 year...

Research paper thumbnail of Promoter methylation of TSC2 gene is important for pathogenesis of TSC and LAM lesions

Research paper thumbnail of Pathogenesis and pharmacological evaluation of human TSC2 smooth muscle cells. Possible treatment options for TSC and lymphangioleiomyomatosis

Tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM) are rare genetic diseases cau... more Tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM) are rare genetic diseases caused by mutations in TSC genes. TSC2-/- cells form hamartomas and invade lungs causing the fatal disease LAM. We have isolated from an angiomyolipoma a homogenous population of TSC2 smooth muscle-like (ASM) cells with LOH genetic lesion (TSC2-/-ASM cells). These cells grow and proliferate in EGF-dependent manner, while auto-secreted IGF-1 acts as a surviving factor. The constitutive phosphorylation of S6, and higher Akt and ERK phosphorylation are a paradigm of these cells. We validated our cells as a cellular model for studying TSC and LAM by TSC2 gene transfection, which promoted the biochemical normalization and eliminated EGF-dependency. The blockade of EGF-receptors with specific antibodies results in TSC2-/- ASM cells death and suggest a novel therapeutic avenue for TSC and LAM. We have recently found that suppression of TSC2 gene may also have an epigenetic origin, another pure ASM cell population was isolated in our lab. These cells showed the typical constitutive S6 phosphorylation, and lacked tuberin as the result of TSC2 promoter methylation as a second hit (TSC2-/methASM cells). Chromatin remodelling agents, such as trichostatin-A and 5-azacytidine de-methylated TSC2-/methASM cell promoter, that returned normal. TSC2-/methASM cells are pharmacologically comparable to TSC2-/-ASM cells. These data show for the first time that TSC2 pathogenesis might be originated also by epigenetic defects in ASM cells. Pulmonary proliferation of TSC2-/-ASM cells in LAM is characterized by cystic destruction of lung parenchyma. To develop a proper animal model for LAM and to validate our in vitro pharmacological observations we have administered by endonasal route TSC2-/-ASM cells to nude mice. TSC2-/-ASM cells massively penetrated into lungs, and from there reached the lymphatic vessels where they accumulated in the lymph nodes. Here these cells grow and proliferate. The progressive increase of TSC2-/-ASM cells in lung parenchyma correspond to the progressive invasion of lymphatic vessel and destruction of lung tissue. Anti-EGFR antibody treatment killed TSC2-/-ASM cells and reversed lung structural alterations, while rapamycin had a weaker effect. These data suggest a novel therapeutic approach for the control of the abnormal ASM cell growth in TSC and lymphangioleiomyomatosi

Research paper thumbnail of Motility and Invasive Properties of Lam/TSC Cells from Chylous of a Lam/TSC Patient

Mutations in TSC2 are common factors in the development of TSC. LAM can occur as a sporadic disor... more Mutations in TSC2 are common factors in the development of TSC. LAM can occur as a sporadic disorder or associated with TSC. We have isolated human TSC2-smooth-muscle-cells from chylous of a LAM/TSC patient (LAM/TSC cells). In these cells tuberin expression is related to a TSC2 epigenetic regulation, and proliferation and survival are EGF-dependent. Cancer metastasis results in altered cell motility and invasiveness and is modulated by Notch activity. Given the fact that LAM/TSC cells express mesenchymal features, and survive in adherent or nonadherent status, we studied their invasive properties and motility. LAM/TSC cells showed anoikis resistence in soft agar assay confirming their loss of contact inhibition and acquisition of anchorage independent survival. Cell motility appear to be related to tuberin expression since it was reduced by 5-azacytidine treatment. Rapamycin and anti-EGFR decreased the migration rate but at a slightly lower extent than 5-azacytidine. Adherent and nonadherent LAM/TSC cells invaded epithelial cell layers by feeder cell invasion assay with an increased of IL-6 and IL-8 secretion. LAM/TSC cells express Notch and its targets (e.i. Hes, Jagged 1,2) that are not regulated by specific inhibitors. In conclusion, LAM/TSC cells express invasive and migratory features which appear to be related to tuberin expression

Research paper thumbnail of Anti-EGFR Antibody Reduces Lung Nodules by Inhibition of EGFR-Pathway in a Model of Lymphangioleiomyomatosis

BioMed Research International, 2015

EGFR belongs to the HER/ErbB family of tyrosine kinase receptors and its activation in cancer cel... more EGFR belongs to the HER/ErbB family of tyrosine kinase receptors and its activation in cancer cells has been linked with increased proliferation, angiogenesis, and metastasis. Lymphangioleiomyomatosis (LAM) is a rare, low-grade neoplasm that occurs sporadically or in association with tuberous sclerosis complex (TSC), a genetic, multisystem disorder characterized by hamartomas in several organs. From chylous of a LAM/TSC patient, we previously isolated smooth muscle-like LAM/TSC cells whose proliferation depends on EGF and monoclonal anti-EGFR antibodies reduced proliferation and caused cell death. We demonstrated that the dependency from EGF was caused by the absence of tuberin. To study the role of EGFR pathwayin vivo, we developed a mouse model by administration of LAM/TSC cells to female nude mice. LAM/TSC cells caused pulmonary airspace enlargement and, after 30 weeks, nodule formation which express EGFR. Anti-EGFR antibody decreased the number and dimension of lung nodules like...

Research paper thumbnail of Experimental diabetic neuropathy, inhibition of excessive protein mono-ADP-ribosylation prevents reduction of substance P axonal transport

Pharmacological Research, 1995

ABSTRACT

Research paper thumbnail of Chromatin Remodeling by Rosuvastatin Normalizes TSC2-/meth Cell Phenotype through the Expression of Tuberin

Journal of Pharmacology and Experimental Therapeutics, 2013

Tuberous sclerosis complex (TSC) is a multi-systemic syndrome caused by mutations in TSC1 or TSC2... more Tuberous sclerosis complex (TSC) is a multi-systemic syndrome caused by mutations in TSC1 or TSC2 gene. In TSC2null cells, Rheb, a member of the Ras family of GTPases, is constitutively activated. Statins inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase and block the synthesis of isoprenoid lipids with inhibition of Rheb farnesylation and RhoA geranylgeranylation. The effects of rosuvastatin on the function of human TSC2 2/2 and TSC2-/meth a-actin smooth muscle (ASM) cells have been investigated. The TSC2 2/2 and TSC2-/meth ASM cells, previously isolated in our laboratory from the renal angiomyolipoma of two TSC patients, do not express tuberin and bear loss of heterozigosity caused by a double hit on TSC2 and methylation of TSC2 promoter, respectively. Exposure to rosuvastatin affected TSC2-/meth ASM cell growth and promoted tuberin expression by acting as a demethylating agent. This occurred without changes in interleukin release. Rosuvastatin also reduced RhoA activation in TSC2-/meth ASM cells, and it required coadministration with the specific mTOR (mammalian target of rapamycin) inhibitor rapamycin to be effective in TSC2 2/2 ASM cells. Rapamycin enhanced rosuvastatin effect in inhibiting cell proliferation in TSC2 2/2 and TSC2-/meth ASM cells. Rosuvastatin alone did not alter phosphorylation of S6 and extracellular signal-regulated kinase (ERK), and at the higher concentration, rosuvastatin and rapamycin slightly decreased ERK phosphorylation. These results suggest that rosuvastatin may potentially represent a treatment adjunct to the therapy with mTOR inhibitors now in clinical development for TSC. In particular, rosuvastatin appears useful when the disease is originated by epigenetic defects.

Research paper thumbnail of Diabetic neuropathy in the rat: 1. Alcar augments the reduced levels and axoplasmic transport of substance P

Journal of Neuroscience Research, 1995

This study examined the sciatic nerve axonal transport of substance P‐like immunoreactivity (SPLI... more This study examined the sciatic nerve axonal transport of substance P‐like immunoreactivity (SPLI) and its basal content in stomach, sciatic nerve and lumbar spinal cord of 8‐ and 12‐week alloxan‐diabetic rats, respectively. One group of diabetic rats received acetyl‐l‐carnitine (ALCAR) throughout the experimental period. Alloxan treatment caused hyperglycemia and reduced body growth. Axonal transport of SPLI was studied by measurement of 24‐hour accumulation at a ligature on the sciatic nerve. There was a marked reduction (from 50% to 100% according to the nerve segment examined) of anterograde and retrograde accumulation of SPLI in the constricted nerve of 8‐week diabetic rats. In the sciatic nerve of ALCAR‐treated diabetic rats, the accumulation of SPLI was comparable to control values. In the sciatic nerve, lumbar spinal cord and stomach of 12‐week diabetic rats, there is a significant reduction of SPLI content. ALCAR treatment prevented SPLI loss in these tissues. Sciatic nerve...

Research paper thumbnail of Perinatal morphine II: Changes in cortical plasticity

Journal of Neuroscience Research, 1995

We have previously shown that perinatal exposure to morphine impairs reactive plasticity of serot... more We have previously shown that perinatal exposure to morphine impairs reactive plasticity of serotonin (5‐HT) neurons following selective neonatal lesion (Gorio et al., J Neurosci Res 34:462–471, 1993). This study shows that morphine inhibits also that the compensatory sprouting of intact axons after partial denervation. Neonatal 6‐OHDA injection causes norepinephrine (NE) depletion in the frontal cortex, which triggers a compensatory increase of dopamine, serotonin (5‐HT), and met‐enkephalin content correlated by the increased density of tyrosine hydroxylase– and 5‐HT–positive axons. In perinatal morphine‐treated rats, no compensatory changes are observed after neonatal 6‐OHDA depletion of NE in the frontal cortex. ©1995 Wiley‐Liss, Inc.

Research paper thumbnail of Inhibition of high glucose-induced protein mono-ADP-ribosylation restores neuritogenesis and sodium-pump activity in SY5Y neuroblastoma cells

Journal of Neuroscience Research, 1999

The exposure of SY5Y neuroblastoma cells to high concentrations of glucose, fructose, or galactos... more The exposure of SY5Y neuroblastoma cells to high concentrations of glucose, fructose, or galactose is an experimental model commonly used for in vitro evaluation of typical neuronal alterations observed in diabetes mellitus. In the present study, we observed that 2 weeks of exposure to high carbohydrate concentrations caused both a significant impairment in neurite formation induced by supplementation of retinoic acid or by subtraction of fetal calf serum to the culture medium and a marked reduction in Na(+)-K(+)-ATPase activity. However, only the exposure to high millimoles of glucose caused an enhancement of mono-ADP-ribosylation, typical of diabetes mellitus, affecting at least five proteins. The concomitant exposure to high glucose and to silybin, a mono-ADP-ribosylation inhibitor, normalized the extent of ADP-ribosylation of the five proteins and counteracted the inhibitory effects of high glucose on Na(+)-pump activity and on neuritogenesis. Conversely, the supplementation of silybin did not prevent fructose and galactose inhibitory effects on Na(+)-pump activity and neurite formation. These data confirm those of previous reports suggesting a link between excessive protein mono-ADP-ribosylation and the onset of diabetic complications such as diabetic neuropathy.

Research paper thumbnail of Glycosaminoglycans treatment increases IGF-I muscle levels and counteracts motor neuron death: A novel nonanticoagulant action

Journal of Neuroscience Research, 1999

The present study shows that sciatic nerve crush in 2-day-old rats causes extensor digitorum long... more The present study shows that sciatic nerve crush in 2-day-old rats causes extensor digitorum longus (EDL) muscle atrophy and motor neuron loss and that treatment with glycosaminoglycans (GAGs) promotes muscle reinnervation, motor neuron survival, and markedly increases insulin-like growth factor-I (IGF-I) content in the denervated muscles. EDL muscle denervation-induced atrophy in saline-treated rats is progressive and reaches the greatest extent at 42 days after birth, which correlates with reduced EDL weight growth. There is also a partial reinnervation as shown by the number of reinnervated EDL muscle fibers (65.4% of control) and by the poor restoration of the indirect isometric twitch tension (62% of control) that is further reduced under tetanic stimulation (34% of control). The number of surviving motor neurons that innervate EDL muscle drops from 55 +/- 3 to 29 +/- 8. In GAGs-treated 42-day-old rats, the effects of neonatal nerve lesioning on EDL muscle atrophy and denervation are successfully reversed, and the isometric twitch tension and the capacity to hold tetanic stimulation are restored to almost control levels. The number of surviving EDL motor neurons is also increased to 43 +/- 4. Treatment with GAGs selectively affects IGF-I content in denervated hindlimb muscles, which is augmented from 7.02 +/- 0.71 ng/mg tissue to 25.72 +/- 0.7 in the EDL and from 3.2 +/- 0.18 to a robust 211 +/- 9.6 in the soleus.

Research paper thumbnail of G protein-mediated signal transduction is affected in primary biliary cirrhosis

Hepatology Research, 2006

Primary biliary cirrhosis (PBC) is a chronic liver disease characterized by severe intraepatic ch... more Primary biliary cirrhosis (PBC) is a chronic liver disease characterized by severe intraepatic cholestasis. Pruritus often occurs during the course of the illness. We designed a study aimed at assessing whether pruritus is associated with dysfunction of signal transduction. Seventeen female patients affected by PBC were enrolled into the study and divided in two groups according to severity of liver disease. Leukocytes were isolated from peripheral blood and Gi, Go and Gs protein expressions were evaluated by western blotting, while G protein function was assessed by measuring cyclic adenosine phosphate formation. The expression of all types of G proteins was increased in leukocytes of PBC patients. The basal adenylate activity was significally higher than control in patients with less severe liver disease, while it was lower than normal in those with severe liver disease. Incubation of patient leukocytes with guanosine triphosphate-gamma-S and Gs protein activators failed to enhance cAMP production, while N-formyl-met-leu-phe was more effective in reducing cAMP production. The expression of all G proteins was non-selectively increased in PBC leukocytes, while adenylate cyclase activity was significantly modified. However, the observed changes in G proteins expression and in adenylate cyclase activity are not related to the presence of pruritus.