Felicia Carotenuto

Hepatocyte Growth Factor Effects on Mesenchymal Stem Cells: Proliferation, Migration, and Differentiation

Hepatocyte growth factor (HGF), a pleiotropic cytokine of mesenchymal origin promoting migration, proliferation, and survival in a wide spectrum of cells, can also modulate different biological responses in stem cells, but the mechanisms involved are not completely understood so far. In this context, we show that short‐term exposure of mesenchymal stem cells (MSCs) to HGF can induce the activation of its cognate Met receptor and the downstream effectors ERK1/2, p38MAPK, and PI3K/Akt, while long‐term exposure to HGF resulted in cytoskeletal rearrangement, cell migration, and marked inhibition of proliferation through the arrest in the G1‐S checkpoint. When added to MSCs, the K252A tyrosine kinase inhibitor prevented HGF‐induced responses. HGFs effect on MSC proliferation was reversed by p38 inhibitor SB203580, while the effects on cell migration were abrogated by PI3K inhibitor Wortmannin, suggesting that HGF acts through different pathways to determine its complex effects on MSCs. Prolonged treatment with HGF induced the expression of cardiac‐specific markers (GATA‐4, MEF2C, TEF1, desmin, α‐MHC, β‐MHC, and nestin) with the concomitant loss of the stem cell markers nucleostemin, c‐kit, and CD105.

Criticality of the Biological and Physical Stimuli Array Inducing Resident Cardiac Stem Cell Determination

The replacement of injured cardiac contractile cells with stem cell‐derived functionally efficient cardiomyocytes has been envisaged as the resolutive treatment for degenerative heart diseases. Nevertheless, many technical issues concerning the optimal procedures to differentiate and engraft stem cells remain to be answered before heart cell therapy could be routinely used in clinical practice. So far, most studies have been focused on evaluating the differentiative potential of different growth factors without considering that only the synergistic cooperation of biochemical, topographic, chemical, and physical factors could induce stem cells to adopt the desired phenotype. The present study demonstrates that the differentiation of cardiac progenitor cells to cardiomyocytes does not occur when cells are challenged with soluble growth factors alone, but requires strictly controlled procedures for the isolation of a progenitor cell population and the artifactual recreation of a microenvironment critically featured by a fine‐tuned combination of specific biological and physical factors. Indeed, the scaffold geometry and stiffness are crucial in enhancing growth factor differentiative effects on progenitor cells. The exploitation of this concept could be essential in setting up suitable procedures to fabricate functionally efficient engineered tissues.

Brain Natriuretic Peptide (BNP) regulates the production of inflammatory mediators in human THP-1 macrophages.

Brain Natriuretic Peptide (BNP), besides retaining vasodilatory, diuretic and natriuretic properties, is a vasoactive hormone that it is also involved in several cardiac diseases as well as severe sepsis and septic shock. All these conditions are characterized by an ongoing inflammatory response consisting in a complex interaction of pleiotropic mediators derived from plasma or cells, including monocytes and macrophages. However, the relationship between this hormone and inflammation remains to be elucidated. Therefore, the aim of the present study was to evaluate a possible BNP immunomodulatory activity on macrophages. Our results demonstrate that BNP regulates the production of major inflammatory molecules, such as reactive oxygen- and nitrogen species (ROS and RNS), leukotriene B(4) (LTB(4)), prostaglandin E(2) (PGE(2)); modulates the cytokines (TNF-alpha, IL-12 and IL-10) profile, and affects cell motility. These results furnish novel and brand-new proofs on BNP ability of modulating the production of inflammatory mediators in macrophages whose role has broad implications in inflammatory states where increased BNP levels have been reported.

Stem cell activation sustains hereditary hypertrophy in hamster cardiomyopathy.

Recent studies have documented the presence of stem cells within the myocardium and their role in the repair of ischaemic injury. Nevertheless, the pathogenic role of stem cells in non‐ischaemic myocardial diseases, as well as the factors potentially responsible for their activation, is still under debate. The present study demonstrates the presence of an increased number of c‐kit positive, MDR‐positive, and Sca‐1‐positive stem cells within the myocardium of hereditary δ‐SG null hamsters, a spontaneously occurring model of hypertrophic cardiomyopathy. When hamsters are 80 days old, ie at the ‘hypertrophic’ stage of the disease, but without haemodynamic overload, these cells associate with a multitude of cells co‐expressing c‐kit, cMet, GATA4, or MEF‐2, and proliferating myocytes co‐expressing myosin heavy chain, telomerase, ki67 and cyclin B. Furthermore, at the same animal age, the number of myocardial cells co‐expressing c‐kit and Flk‐1, and the number of capillary vessels, is also amplified. In order to identify factors potentially responsible for stem cell activation, the myocardial expression of HGF and cMet and HGF plasma levels were evaluated, demonstrating their increase in 80‐day‐old δ‐SG null hamsters. To demonstrate the possible ability of HGF to induce stem cell differentiation, bone‐marrow‐derived mesenchymal stem cells were challenged with HGF at the same plasma concentration observed in vivo. HGF induced cMet phosphorylation, and caused loss of stem cell features and overexpression of MEF‐2, TEF1, and MHC. Our results demonstrate that stem cell activation occurs within the cardiomyopathic myocardium, very likely to maintain an efficient cardiac architecture. In this context, elevated levels of HGF might play a role in induction of stem cell commitment to the cardiomyocyte lineage and in cardioprotection through its anti‐apoptotic action. Consistently, when cytokine levels declined to physiological concentrations, as in 150‐day‐old cardiomyopathic animals, myocardial apoptosis prevailed, prejudicing cardiac function. Copyright

Thick Soft Tissue Reconstruction on Highly Perfusive Biodegradable Scaffolds

The lack of a vascular network and poor perfusion is what mostly prevents three-dimensional (3D) scaffolds from being used in organ repair when reconstruction of thick tissues is needed. Highly-porous scaffolds made of poly(L-lactic acid) (PLLA) are prepared by directional thermally induced phase separation (dTIPS) starting from 1,4-dioxane/PLLA solutions. The influence of polymer concentration and temperature gradient, in terms of imposed intensity and direction, on pore size and distribution is studied by comparison with scaffolds prepared by isotropic TIPS. The processing parameters are optimized to achieve an overall porosity for the 3D scaffolds of about 93% with a degree of interconnectivity of 91%. The resulting pore network is characterized by the ordered repetition of closely packed dendrite-like cavities, each one showing stacks of 20 microm large side lamellar branches departing from 70 microm diameter vertical backbones, strongly resembling the vascular patterns. The in vitro biological responses after 1 and 2 weeks are evaluated from mesenchymal (bone marrow stromal) cells (MSC) static culturing. A novel vacuum-based deep-seeding method is set up to improve uniform cell penetration down to scaffold thicknesses of over 1 mm. Biological screenings show significant 3D scaffold colonization even after 18 h, while cellular retention is observed up to 14 d in vitro (DIV). Pore architecture-driven cellular growth is accompanied by cell tendency to preserve their multi-potency towards differentiation. Confluent tissues as thick as 1 mm were reconstructed taking advantage of the large perfusion enhanced by the highly porous microstructure of the engineered scaffolds, which could successfully serve for applications aimed at vascular nets and angiogenesis.

Tuning hierarchical architecture of 3D polymeric scaffolds for cardiac tissue engineering

Tissue engineering combines the fields of engineering, chemistry, biology, and medicine to fabricate replacement tissues able to restore, maintain, or improve structurally and functionally damaged organs. The approach of regenerative medicine is of paramount importance for treating patients with severe cardiac diseases. For successful exploitation, the challenge for cardiac regenerative medicine is to identify the suitable combination between the best cell source for cardiac repair and the design of the optimal scaffold as a template for tissue replacement. Adult stem cells have the potential to improve regenerative medicine with their peculiar feature to self-renew and differentiate into various phenotypes. Insights into the stem cell field lead to the identification of the suitable scaffold features that enhance the ex vivo proliferation and differentiation of stem cells. Scaffolds composed of natural and/or synthetic polymers can organise stem cells into complex architectures that mimic native tissues. To achieve this, a proper design of the chemical, mechanical, and morphological characteristics of the scaffold at different length scales is needed to reproduce the tissue complexity at the cell-scaffold interface. Hierarchical porosities are needed in a single construct, at the millimetre scale to help nutrition and vascularisation, at the micrometer scale to accommodate cells, and at the nanometre scale to favour the expression of extra-cellular matrix components. The present study has been undertaken to setup strategies to integrate stem cells and tailored scaffolds, as a tool to control cardiac tissue regeneration. Among the many available techniques for scaffold fabrication, porogen leaching, phase separation, and electrospinning were selected as low-cost and user-friendly technologies to fabricate tuneable, hierarchically porous matrices that mimic aspects of the cell native surroundings. The biological validation of these scaffolds was performed by implanting adult stem cells.

An omega-3 fatty acid-enriched diet prevents skeletal muscle lesions in a hamster model of dystrophy.

Currently, despite well-known mutational causes, a universal treatment for neuromuscular disorders is still lacking, and current therapeutic efforts are mainly restricted to symptomatic treatments. In the present study, δ-sarcoglycan-null dystrophic hamsters were fed a diet enriched in flaxseed-derived ω3 α-linolenic fatty acid from weaning until death. α-linolenic fatty acid precluded the dystrophic degeneration of muscle morphology and function. In fact, in dystrophic animals fed flaxseed-derived α-linolenic fatty acid, the histological appearance of the muscular tissue was improved, the proliferation of interstitial cells was decreased, and the myogenic differentiation originated new myocytes to repair the injured muscle. In addition, muscle myofibers were larger and cell membrane integrity was preserved, as witnessed by the correct localization of α-, β-, and γ-sarcoglycans and α-dystroglycan. Furthermore, the cytoplasmic accumulation of both β-catenin and caveolin-3 was abolished in dystrophic hamster muscle fed α-linolenic fatty acid versus control animals fed standard diet, while α-myosin heavy chain was expressed at nearly physiological levels. These findings, obtained by dietary intervention only, introduce a novel concept that provides evidence that the modulation of the plasmalemma lipid profile could represent an efficacious strategy to ameliorate human muscular dystrophy.

A diet supplemented with ALA-rich flaxseed prevents cardiomyocyte apoptosis by regulating caveolin-3 expression

AIMSnn-3 polyunsaturated fatty acids (PUFAs) induce beneficial effects on the heart, but the mechanisms through which these effects are operated are not completely clarified yet. Among others, cardiac diseases are often associated with increased levels of cytokines, such as tumour necrosis factor-α (TNF), that cause degeneration and death of cardiomyocytes. The present study has been carried out to investigate (i) the potential anti-apoptotic effects induced by the n-3 polyunsaturated α-linolenic acid (ALA) in experimental models of cardiac diseases characterized by high levels of TNF, and (ii) the potential role of caveolin-3 (Cav-3) in the mechanisms involved in this process.nnnMETHODS AND RESULTSnAn ALA-rich flaxseed diet, administered from weaning to hereditary cardiomyopathic hamsters, prevented the onset of myocardial apoptosis associated with high plasma and tissue levels of TNF preserving caveolin-3 expression. To confirm these findings, isolated neonatal mouse cardiomyocytes were exposed to TNF to induce apoptosis. ALA pre-treatment greatly enhanced Cav-3 expression hampering the internalization of the caveolar TNF receptor and, thus, determining the abortion of the apoptotic vs. survival cascade.nnnCONCLUSIONnThis study unveiled the Cav-3 pivotal role in defending cardiomyocytes against the TNF pro-apoptotic action and the ALA capacity to regulate this mechanism preventing cardiac degenerative diseases.

Activatory Properties of Lysophosphatidic Acid on Human THP-1 Cells

Excessive leukocyte proliferation and proinflammatory mediators release represent common phenomena in several chronic inflammatory diseases. Multiple evidences identify lysophosphatidic acid (LPA), a small lipid endowed with pleiotropic activities, as an important modulator of both proliferation and activation of different cell types involved in several inflammation-associated pathologies. However, its possible role on monocyte proinflammatory activation is not fully understood yet. Aim of the present study was to investigate LPA effects on THP-1 cells in terms of proliferation, reactive oxygen intermediates (ROI) production and release of arachidonic acid-derived inflammatory mediators. Actually, LPA significantly increased both DNA synthesis and ROI production as well as prostaglandin E2 release and the upregulation of LPA3 receptor expression. These findings identified LPA as both a growth factor and a triggering mediator of proinflammatory response in THP-1 cells.

Identification of a new missense mutation in the mtDNA of hereditary hypertrophic, but not dilated cardiomyopathic hamsters

The cardiomyopathic hamster is characterized by a naturally occurring deletion in the δ-sarcoglycan gene generating either the hypertrophic or the dilatative phenotype of cardiomyopathy. This evidence suggests that other genetic or environmental factors might concur to the pathogenesis of cardiomyopathy. The aim of the present study was to investigate on the possibility that other genes are involved in the pathogenesis of hamster cardiomyopathy. For this purpose, a series of genes of cardiomyopathic and healthy hamsters were compared by the differential display technique. The hamster cytochrome c oxidase mitochondrial subunit III (COIII) gene has been sequenced and identified as the gene upregulated in brain and skeletal muscle. The gene sequencing and restriction analysis demonstrated that a missense mutation is present in the COIII gene of hamsters exhibiting hypertrophic cardiomyopathy while no mutations were present in dilatative cardiomyopathic hamsters. The mutation was heteroplasmic and the heteroplasmy level was increased with age in skeletal muscle and heart. The ultrastructural analysis of cardiac tissue showed severe damage in the mitochondrial structure of hypertrophic but not dilatative hamster hearts. These results suggest that the pathogenesis of the cardiac damage in hypertrophic cardiomyopathic hamster may be sustained by multiple mutations exerting a cumulative effect on both structure and function of cardiac muscle.