Michael Del Tatto

Space travel directly induces skeletal muscle atrophy

Space travel causes rapid and pronounced skeletal muscle wasting in humans that reduces their long‐term flight capabilities. To develop effective countermeasures, the basis of this atrophy needs to be better understood. Space travel may cause muscle atrophy indirectly by altering circulating levels of factors such as growth hormone, glucocorticoids, and anabolic steroids and/or by a direct effect on the muscle fibers themselves. To determine whether skeletal muscle cells are directly affected by space travel, tissue‐cultured avian skeletal muscle cells were tissue engineered into bioartificial muscles and flown in perfusion bioreactors for 9 to 10 days aboard the Space Transportation System (STS, i.e., Space Shuttle). Significant muscle fiber atrophy occurred due to a decrease in protein synthesis rates without alterations in protein degradation. Return of the muscle cells to Earth stimulated protein synthesis rates of both muscle‐specific and extracellular matrix proteins relative to ground controls. These results show for the first time that skeletal muscle fibers are directly responsive to space travel and should be a target for countermeasure development.—Vandenburgh, H., Chromiak, J., Shansky, J., Del Tatto, M., Lemaire, J. Space travel directly induces skeletal muscle atrophy. FASEB J. 13, 1031–1038 (1999)

Tissue-Engineered Human Bioartificial Muscles Expressing a Foreign Recombinant Protein for Gene Therapy

Murine skeletal muscle cells transduced with foreign genes and tissue engineered in vitro into bioartificial muscles (BAMs) are capable of long-term delivery of soluble growth factors when implanted into syngeneic mice (Vandenburgh et al., 1996b). With the goal of developing a therapeutic cell-based protein delivery system for humans, similar genetic tissue-engineering techniques were designed for human skeletal muscle stem cells. Stem cell myoblasts were isolated, cloned, and expanded in vitro from biopsied healthy adult (mean age, 42 +/- 2 years), and elderly congestive heart failure patient (mean age, 76 +/- 1 years) skeletal muscle. Total cell yield varied widely between biopsies (50 to 672 per 100 mg of tissue, N = 10), but was not significantly different between the two patient groups. Percent myoblasts per biopsy (73 +/- 6%), number of myoblast doublings prior to senescence in vitro (37 +/- 2), and myoblast doubling time (27 +/- 1 hr) were also not significantly different between the two patient groups. Fusion kinetics of the myoblasts were similar for the two groups after 20-22 doublings (74 +/- 2% myoblast fusion) when the biopsy samples had been expanded to 1 to 2 billion muscle cells, a number acceptable for human gene therapy use. The myoblasts from the two groups could be equally transduced ex vivo with replication-deficient retroviral expression vectors to secrete 0.5 to 2 microg of a foreign protein (recombinant human growth hormone, rhGH)/10(6) cells/day, and tissue engineered into human BAMs containing parallel arrays of differentiated, postmitotic myofibers. This work suggests that autologous human skeletal myoblasts from a potential patient population can be isolated, genetically modified to secrete foreign proteins, and tissue engineered into implantable living protein secretory devices for therapeutic use.

A simplified method for tissue engineering skeletal muscle organoids in vitro

Tissue-engineered three dimensional skeletal muscle organ-like structures have been formed in vitro from primary myoblasts by several different techniques. This report describes a simplified method for generating large numbers of muscle organoids from either primary embryonic avian or neonatal rodent myoblasts, which avoids the requirements for stretching and other mechanical stimulation.

Exosomes induce and reverse monocrotaline-induced pulmonary hypertension in mice

AIMS Extracellular vesicles (EVs) from mice with monocrotaline (MCT)-induced pulmonary hypertension (PH) induce PH in healthy mice, and the exosomes (EXO) fraction of EVs from mesenchymal stem cells (MSCs) can blunt the development of hypoxic PH. We sought to determine whether the EXO fraction of EVs is responsible for modulating pulmonary vascular responses and whether differences in EXO-miR content explains the differential effects of EXOs from MSCs and mice with MCT-PH. METHODS AND RESULTS Plasma, lung EVs from MCT-PH, and control mice were divided into EXO (exosome), microvesicle (MV) fractions and injected into healthy mice. EVs from MSCs were divided into EXO, MV fractions and injected into MCT-treated mice. PH was assessed by right ventricle-to-left ventricle + septum (RV/LV + S) ratio and pulmonary arterial wall thickness-to-diameter (WT/D) ratio. miR microarray analyses were also performed on all EXO populations. EXOs but not MVs from MCT-injured mice increased RV/LV + S, WT/D ratios in healthy mice. MSC-EXOs prevented any increase in RV/LV + S, WT/D ratios when given at the time of MCT injection and reversed the increase in these ratios when given after MCT administration. EXOs from MCT-injured mice and patients with idiopathic pulmonary arterial hypertension (IPAH) contained increased levels of miRs-19b,-20a,-20b, and -145, whereas miRs isolated from MSC-EXOs had increased levels of anti-inflammatory, anti-proliferative miRs including miRs-34a,-122,-124, and -127. CONCLUSION These findings suggest that circulating or MSC-EXOs may modulate pulmonary hypertensive effects based on their miR cargo. The ability of MSC-EXOs to reverse MCT-PH offers a promising potential target for new PAH therapies.

Microvesicle Induction of Prostate Specific Gene Expression in Normal Human Bone Marrow Cells

PURPOSE Transfer of genetic material from cancer cells to normal cells occurs via microvesicles. Cell specific phenotypes can be induced in normal cells by the transfer of material in microvesicles, leading to genetic changes. We report the identification and expression of prostate specific genes in normal human marrow cells co-cultured with human prostate cancer cells. MATERIALS AND METHODS We harvested prostate tissue from 11 patients with prostate cancer. In 4 cases prostate tissue was co-cultured across from human marrow for 2 or 7 days but separated from it by a 0.4 μM polystyrene membrane. In 5 cases conditioned medium from patient cancer tissue was collected and ultracentrifuged, and microvesicles were collected for co-culture (3) and vesicle characterization (3). Explanted human marrow was harvested from cultures and RNA extracted. Real-time reverse transcriptase-polymerase chain reaction was done for select prostate specific genes. RESULTS Marrow exposed to human prostate tumor or isolated microvesicles in culture in 4 and 3 cases, respectively, showed at least 2-fold or greater prostate gene expression than control marrow. In 1 case in which normal prostate was co-cultured there were no prostate gene increases in normal marrow. CONCLUSIONS Prostate cancer tumor cells co-cultured with human bone marrow cells induce prostate specific gene expression. The proposed mechanism of transfer of genetic material is via microvesicles. This represents an opportunity for novel therapeutic agents, such as antibodies, to block microvesicle release from cancer cells or for agents that may block cells from accepting microvesicles.

Marrow cell genetic phenotype change induced by human lung cancer cells.

Microvesicles have been shown to mediate varieties of intercellular communication. Work in murine species has shown that lung-derived microvesicles can deliver mRNA, transcription factors, and microRNA to marrow cells and alter their phenotype. The present studies evaluated the capacity of excised human lung cancer cells to change the genetic phenotype of human marrow cells. We present the first studies on microvesicle production by excised cancers from human lung and the capacity of these microvesicles to alter the genetic phenotype of normal human marrow cells. We studied 12 cancers involving the lung and assessed nine lung-specific mRNA species (aquaporin, surfactant families, and clara cell-specific protein) in marrow cells exposed to tissue in co-culture, cultured in conditioned media, or exposed to isolated lung cancer-derived microvesicles. We assessed two or seven days of co-culture and marrow which was unseparated, separated by ficoll density gradient centrifugation or ammonium chloride lysis. Under these varying conditions, each cancer derived from lung mediated marrow expression of between one and seven lung-specific genes. Microvesicles were identified in the pellet of ultracentrifuged conditioned media and shown to enter marrow cells and induce lung-specific mRNA expression in marrow. A lung melanoma and a sarcoma also induced lung-specific mRNA in marrow cells. These data indicate that lung cancer cells may alter the genetic phenotype of normal cells and suggest that such perturbations might play a role in tumor progression, tumor recurrence, or metastases. They also suggest that the tissue environment may alter cancer cell gene expression.

Organogenesis of skeletal muscle in tissue culture.

Skeletal muscle structure is regulated by many factors, including nutrition, hormones, electrical activity, and tension. The muscle cells are subjected to both passive and active mechanical forces at all stages of development, and these forces play important but poorly understood roles in regulating muscle organogenesis and growth. For example, during embryogenesis, the rapidly growing skeleton places large passive mechanical forces on the attached muscle tissue. These forces not only help to organize the proliferating mononucleated myoblasts into the oriented, multinucleated myofibers of a functional muscle, but also tightly couple the growth rate of muscle to that of bone. Postnatally, the actively contracting, innervated muscle fibers are subjected to different patterns of active and passive tensions that regulate longitudinal and cross-sectional myofiber growth. These mechanically induced organogenic processes have been difficult to study under normal tissue culture conditions, resulting in the development of numerous methods and specialized equipment to simulate the in vivo mechanical environment (1-4). These techniques have led to the engineering of bioartificial muscles (organoids), which display many of the characteristics of in vivo muscle, including parallel arrays of postmitotic fibers organized into fascicle-like structures with tendon-like ends. They are contractile, express adult isoforms of contractile proteins, perform directed work, and can be maintained in culture for long periods.

Reversal of chemosensitivity and induction of cell malignancy of a non-malignant prostate cancer cell line upon extracellular vesicle exposure

BackgroundExtracellular vesicle (EV) trafficking is a fundamental cellular process that occurs in cells and is required for different aspects of pathophysiology. EV trafficking leads to changes in cellular function including apoptosis, angiogenesis and proliferation required for increased tumor formation.ResultsWe report several phenotypic changes mediated by EVs isolated from non-malignant and malignant prostate cells as well as patient biopsied prostate tumor samples. EVs can reverse the resistance of prostate cancer cells to camptothecin EVs isolated from non-malignant PrECs (Prostate Epithelial Cells) can reverse soft agar colony formation of malignant DU145 cells, with the reciprocal effect observed. Isolation of EVs from 2 Gleason grade 8 prostate cancer patients significantly induced soft agar colony formation of non-malignant PrECs. We have identified proteins via antibody and Mass spectrometry analysis that may be responsible for the phenotypic changes. Mass spectrometry analysis of protein lysates using ProteoIQ revealed protein candidates associated with gene ontology annotations that may be responsible for this phenotypic change. Ingenuity Pathway Analysis was used to identify statistically relevant canonical pathways and functions associated the protein IDs and expression values obtained using ProteoIQ. Western blot analysis confirmed the increase of 14-3-3 zeta, pRKIP and prohibitin protein levels in PrEC cells co-cultured with patient EVs. 14-3-3 proteins were also found as common proteins of 3 other Gleason grade 8 patients.ConclusionOur study provides a rational basis to further investigate putative proteins, such as 14-3-3 and prohibitin and genetic factors that may be responsible for phenotypic changes that are associated with prostate cancer progression.

Extracellular vesicle-mediated phenotype switching in malignant and non-malignant colon cells

BackgroundExtracellular vesicles (EVs) are secreted from many cells, carrying cargoes including proteins and nucleic acids. Research has shown that EVs play a role in a variety of biological processes including immunity, bone formation and recently they have been implicated in promotion of a metastatic phenotype.MethodsEVs were isolated from HCT116 colon cancer cells, 1459 non-malignant colon fibroblast cells, and tumor and normal colon tissue from a patient sample. Co-cultures were performed with 1459 cells and malignant vesicles, as well as HCT116 cells and non-malignant vesicles. Malignant phenotype was measured using soft agar colony formation assay. Co-cultures were also analyzed for protein levels using mass spectrometry. The importance of 14-3-3 zeta/delta in transfer of malignant phenotype was explored using siRNA. Additionally, luciferase reporter assay was used to measure the transcriptional activity of NF-κB.ResultsThis study demonstrates the ability of EVs derived from malignant colon cancer cell line and malignant patient tissue to induce the malignant phenotype in non-malignant colon cells. Similarly, EVs derived from non-malignant colon cell lines and normal patient tissue reversed the malignant phenotype of HCT116 cells. Cells expressing an EV-induced malignant phenotype showed increased transcriptional activity of NF-κB which was inhibited by the NF--κB inhibitor, BAY117082. We also demonstrate that knock down of 14-3-3 zeta/delta reduced anchorage-independent growth of HCT116 cells and 1459 cells co-cultured with HCT derived EVs.ConclusionsEvidence of EV-mediated induction of malignant phenotype, and reversal of malignant phenotype, provides rational basis for further study of the role of EVs in tumorigenesis. Identification of 14-3-3 zeta/delta as up-regulated in malignancy suggests its potential as a putative drug target for the treatment of colorectal cancer.

Expression of Cell Cycle-Related Genes With Cytokine-Induced Cell Cycle Progression of Primitive Hematopoietic Stem Cells

Primitive marrow lineage-negative rhodamine low and Hoechst low (LRH) stem cells isolated on the basis of quiescence respond to the cytokines thrombopoietin, FLT3L, and steel factor by synchronously progressing through cell cycle. We have now profiled the mRNA expression, as determined by real-time RT-PCR, of 47 hematopoietic or cell cycle-related genes, focusing on the variations in the cell cycle regulators with cycle transit. LRH stem cells, at isolation, showed expression of all interrogated genes, but at relatively low levels. In our studies, there was a good deal of consistency with regard to cell cycle regulatory genes involved in the G1/S progression point of LRH murine stem cells. The observed pattern of expression of cyclin A2 is consistent with actions at these phases of cell cycle. Minimal elevations were seen at 16 h with higher elevations at 24, 32, 40, and 48 h times encompassing S, G2, and M phases. CDK2 expression pattern was also consistent with a role in G1/S transition with a modest elevation at 24 h and more substantial elevation at 32 h. The observed pattern of expression of cyclin F mRNA with marked elevations at 16-40 h was also consistent with actions in S and G2 phases. Cyclin D1 expression pattern was less consistent with its known role in G1 progression. The alterations in multiple other cell cycle regulators were consistent with previous information obtained in other cell systems. The cycle regulatory mechanics appears to be preserved across broad ranges of cell types.