Regulation of long-distance transport of mitochondria along microtubules

Mitochondria are cellular organelles of crucial importance, playing roles in cellular life and death. In certain cell types, such as neurons, mitochondria must travel long distances so as to meet metabolic demands of the cell. Mitochondrial movement is essentially microtubule (MT) based and is executed by two main motor proteins, Dynein and Kinesin. The organization of the cellular MT network and the identity of motors dictate mitochondrial transport. Tight coupling between MTs, motors, and the mitochondria is needed for the organelle precise localization. Two adaptor proteins are involved directly in mitochondria-motor coupling, namely Milton known also as TRAK, which is the motor adaptor, and Miro, which is the mitochondrial protein. Here, we discuss the active mitochondria transport process, as well as motor–mitochondria coupling in the context of MT organization in different cell types. We focus on mitochondrial trafficking in different cell types, specifically neurons, migrating cells, and polarized epithelial cells.     

De novo hem- and lymphangiogenesis by endothelial progenitor and mesenchymal stem cells in immunocompetent mice

Cellular pro-angiogenic therapies may be applicable for the treatment of peripheral vascular diseases. Interactions between mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) may provide such a treatment option. With the exception of some studies in man, experiments have only been performed in immunodeficient mice and rats. We studied an immunocompetent syngeneic mouse model. We isolated MSCs from bone marrow and EPCs from the lung of adult C57/Bl.6 mice and co-injected them in Matrigel subcutaneously in adult C57/Bl.6 mice. We demonstrate development of both blood vessels and lymphatics. Grafted EPCs integrated into the lining of the two vessel types, whereas MSCs usually did not incorporate into the vessel wall. Injections of each separate cell type did not, or hardly, reveal de novo angiogenesis. The release of VEGF-A by MSCs has been shown before, but its inhibitors, e.g., soluble VEGF receptors, have not been studied. We performed qualitative and quantitative studies of the proteins released by EPCs, MSCs, and cocultures of the cells. Despite the secretion of VEGF inhibitors (sVEGFR-1, sVEGFR-2) by EPCs, VEGF-A was secreted by MSCs at bioavailable amounts (350 pg/ml). We confirm the secretion of PlGF, FGF-1, MCP-1, and PDGFs by EPCs/MSCs and suggest functions for VEGF-B, amphiregulin, fractalkine, CXCL10, and CXCL16 during MSC-induced hem- and lymphangiogenesis. We assume that lymphangiogenesis is induced indirectly by growth factors from immigrating leukocytes, which we found in close association with the lymphatic networks. Inflammatory responses to the cellular markers GFP and cell-tracker red (CMPTX) used for tracing of EPCs or MSCs were not observed. Our studies demonstrate the feasibility of pro-angiogenic/lymphangiogenic therapies in immunocompetent animals and indicate new MSC/EPC-derived angiogenic factors.     

Morphofunctional changes in the mitochondrial subpopulations of conceptus tissues during the placentation process

To establish the role of mitochondrial subpopulations in the mitochondrial maturation process, we studied morphological and functional changes in the mitochondria of different mammalian conceptus tissues during the organogenic and the placentation processes. Mitochondrial subpopulations of three different conceptus tissues, embryo and visceral yolk sac placenta on gestational days 11, 12 and 13 and placenta on days 12 and 13, were examined morphologically by transmission electron microscopy. Cytochrome oxidase activity and protein levels were also measured in each mitochondrial subpopulation. The results indicate two different mitochondrial subpopulation profiles: a homogeneous one, which corresponds to immature mitochondria, and a heterogeneous one, which represents the mature mitochondria. The three tissues studied show different morphologic and metabolic patterns of mitochondrial maturation during the placentation process, rendering them suitable as experimental models to establish the p ossible relationship between mitochondrial maturation and the mitochondrial subpopulations. Received 5 August 2002; received after revision 23 September 2002; accepted 8 October 2002 RID="*" ID="*"Corresponding author.     

Platelet-derived growth factor receptor beta identifies mesenchymal stem cells with enhanced engraftment to tissue injury and pro-angiogenic property

Mesenchymal stem cells (MSCs) are heterogeneous likely consisting of subpopulations with various therapeutic potentials. Here we attempted to acquire a subset of MSCs with enhanced effect in wound healing. We found that human placental MSCs expressing platelet-derived growth factor (PDGF) receptor (PDGFR)-β exhibited greater proliferation rates and generated more colony-forming unit-fibroblast (CFU-F), compared to PDGFR-β^? MSCs. Notably, PDGFR-β⁺ MSCs expressed higher levels of pro-angiogenic factors such as Ang1, Ang2, VEGF, bFGF and PDGF. When 10⁶ GFP-expressing MSCs were topically applied into excisional wounds in mice, PDGFR-β⁺ MSCs actively incorporated into the wound tissue, resulting in enhanced engraftment (3.92 ± 0.31 × 10⁵ remained in wound by 7 days) and accelerated wound closure; meanwhile, PDGFR-β^? MSCs tended to remain on the top of the wound bed with significantly fewer cells (2.46 ± 0.26 × 10⁵) engrafted into the wound, suggesting enhanced chemotactic migration and engraftment of PDGFR-β⁺ MSCs into the wound. Real-Time PCR and immunostain analyses revealed that the expression of PDGF-B was upregulated after wounding; transwell migration assay showed that PDGFR-β⁺ MSCs migrated eightfold more than PDGFR-β^? MSCs toward PDGF-BB. Intriguingly, PDGFR-β⁺ MSC-treated wounds showed significantly enhanced angiogenesis compared to PDGFR-β^? MSC- or vehicle-treated wounds. Thus, our results indicate that PDGFR-β identifies a subset of MSCs with enhanced chemotactic migration to wound injury and effect in promoting angiogenesis and wound healing, implying a greater therapeutic potential for certain diseases.     

Natural history of mesenchymal stem cells,from vessel walls to culture vessels

Mesenchymal stem/stromal cells (MSCs) can regenerate tissues by direct differentiation or indirectly by stimulating angiogenesis, limiting inflammation, and recruiting tissue-specific progenitor cells. MSCs emerge and multiply in long-term cultures of total cells from the bone marrow or multiple other organs. Such a derivation in vitro is simple and convenient, hence popular, but has long precluded understanding of the native identity, tissue distribution, frequency, and natural role of MSCs, which have been defined and validated exclusively in terms of surface marker expression and developmental potential in culture into bone, cartilage, and fat. Such simple, widely accepted criteria uniformly typify MSCs, even though some differences in potential exist, depending on tissue sources. Combined immunohistochemistry, flow cytometry, and cell culture have allowed tracking the artifactual cultured mesenchymal stem/stromal cells back to perivascular anatomical regions. Presently, both pericytes enveloping microvessels and adventitial cells surrounding larger arteries and veins have been described as possible MSC forerunners. While such a vascular association would explain why MSCs have been isolated from virtually all tissues tested, the origin of the MSCs grown from umbilical cord blood remains unknown. In fact, most aspects of the biology of perivascular MSCs are still obscure, from the emergence of these cells in the embryo to the molecular control of their activity in adult tissues. Such dark areas have not compromised intents to use these cells in clinical settings though, in which purified perivascular cells already exhibit decisive advantages over conventional MSCs, including purity, thorough characterization and, principally, total independence from in vitro culture. A growing body of experimental data is currently paving the way to the medical usage of autologous sorted perivascular cells for indications in which MSCs have been previously contemplated or actually used, such as bone regeneration and cardiovascular tissue repair.     

Response of vascular mesenchymal stem/progenitor cells to hyperlipidemia

Hyperlipidemia is a risk factor for atherosclerosis that is characterized by lipid accumulation, inflammatory cell infiltration, and smooth muscle cell proliferation. It is well known that hyperlipidemia is a stimulator for endothelial dysfunction and smooth muscle cell migration during vascular disease development. Recently, it was found that vessel wall contains a variable number of mesenchymal stem cells (MSCs) that are quiescent in physiological conditions, but can be activated by a variety of stimuli, e.g., increased lipid level or hyperlipidemia. Vascular MSCs displayed characteristics of stem cells which can differentiate into several types of cells, e.g., smooth muscle cells, adipocytic, chondrocytic, and osteocytic lineages. In vitro, lipid loading can induce MSC migration and chemokines secretion. After MSC migration into the intima, they play an essential role in inflammatory response and cell accumulation during the initiation and progression of atherosclerosis. In addition, MSC transplantation has been explored as a therapeutic approach to treat atherosclerosis in animal models. In this review, we aim to summarize current progress in characterizing the identity of vascular MSCs and to discuss the mechanisms involved in the response of vascular stem/progenitor cells to lipid loading, as well as to explore therapeutic strategies for vascular diseases and shed new light on regenerative medicine.     

Modulation of hepatic mitochondrial energy efficiency with age

This study was designed to examine the effect of youth-adulthood transition on hepatic mitochondrial energy efficiency. The changes in basal and palmitate-induced proton leak, which contribute to mitochondrial efficiency, were evaluated in mitochondria isolated from the liver of young and adult rats. Alterations in mitochondrial cytochrome oxidase and aconitase specific activities, and in adenine nucleotide translocator content were also assessed. There was no difference in basal proton leak or thermodynamic coupling and efficiency of oxidative phosphorylation in liver mitochondria between the two rat groups. On the other hand, palmitate-induced proton leak increased significantly in adult rats. The function of this uncoupling could be avoidance of elevated formation of reactive oxygen species, which are known to accelerate ageing.Received 17 February 2004; received after revision 30 March 2004; accepted 1 April 2004     

Role of nitric oxide in the functional response to ischemia-reperfusion of heart mitochondria from hyperthyroid rats

We investigated the role of nitric oxide (NO) in the mitochondrial derangement associated with the functional response to ischemia-reperfusion of hyperthyroid rat hearts. Mitochondria were isolated at 3000 g from hearts subjected to ischemia-reperfusion, with or without N-nitro-L-arginine (L-NNA, an NO synthase inhibitor). During reperfusion, hyperthyroid hearts displayed tachycardia and low functional recovery. Their mitochondria exhibited O₂ consumption similar to euthyroid controls, while H₂O₂ production, hydroperoxide, protein-bound carbonyl and nitrotyrosine levels, and susceptibility to swelling were higher. L-NNA blocked the reperfusion tachycardic response and increased inotropic recovery in hyperthyroid hearts. L-NNA decreased mitochondrial H₂O₂ production and oxidative damage, and increased respiration and tolerance to swelling. Such effects were higher in hyperthyroid preparations. These results confirm the role of mitochondria in ischemia-reperfusion damage, and strongly suggest that NO overproduction is involved in the high mitochondrial dysfunction and the low recovery of hyperthyroid hearts from ischemia-reperfusion. L-NNA also decreased protein content and cytochrome oxidase activity of a mitochondrial fraction isolated at 8000 g. This and previous results suggest that the above fraction contains, together with light mitochondria, damaged mitochondria coming from the heaviest fraction, which has the highest cytochrome oxidase activity and capacity to produce H₂O₂. Therefore, we propose that the high mitochondrial susceptibility to swelling, favoring mitochondrial population purification from H₂O₂-overproducing mitochondria, limits hyperthyroid heart oxidative stress.Received 24 March 2004; received after revision 9 June 2004; accepted 5 July 2004     

Spermatozoa: models for studying regulatory aspects of energy metabolism

Spermatozoa are highly specialized cells, and they offer advantages for studying several basic aspects of metabolic control such as the role of adenosine triphosphate-(ATP)-homeostasis for cell function, the mechanisms of fatigue and metabolic depression, the metabolic channelling through the cytoplasm and the organization and regulation of glycolytic enzymes. Spermatozoa of four species with different reproductive modes are, introduced and the first results are presented: Spermatozoa of the marine wormArenicola marina are well adapted to external fertilization in sea water with fluctuating oxygen tension: they are motile for several hours in oxygen-free sea water, even when the ATP level is dramatically reduced. Anaerobic ATP production occurs by alanine, acetate and propionate fermentation probably by the same pathways known from somatic cells of this species. Under aerobic conditions the phosphagen system might function like a shuttle for energy-rich phosphate from mitochondria to the dynein-ATPases. Storage of turkey and carp spermatozoa for several hours without exogenous substrates and oxygen results in the degradation of phosphocreatine and ATP to inorganic phosphate and adenosine monophosphate (AMP), respectively. Despite low energy charges, stored spermatozoa of both species are capable of progressive movements. In carp spermatozoa fatigue of motility is not accompanied by the dramatic acidosis one discusses as an important effect in muscle fatigue. Energy metabolism of boar spermatozoa is typically based on glycolysis consuming extracellular carbohydrates and producing lactate and protons. The sperm seem to tolerate low intracellular pH (<6.5). The lack of a phosphagen system (no energy shuttle from mitochondria to the distal dynein-ATPases) is probably compensated by a high glycolytic ATP-production in the mitochondria-free piece of the flagellum.     

Parvalbumin alters mitochondrial dynamics and affects cell morphology

The Ca²⁺-binding protein parvalbumin (PV) and mitochondria play important roles in Ca²⁺ signaling, buffering and sequestration. Antagonistic regulation of PV and mitochondrial volume is observed in in vitro and in vivo model systems. Changes in mitochondrial morphology, mitochondrial volume and dynamics (fusion, fission, mitophagy) resulting from modulation of PV were investigated in MDCK epithelial cells with stable overexpression/downregulation of PV. Increased PV levels resulted in smaller, roundish cells and shorter mitochondria, the latter phenomenon related to reduced fusion rates and decreased expression of genes involved in mitochondrial fusion. PV-overexpressing cells displayed increased mitophagy, a likely cause for the decreased mitochondrial volumes and the smaller overall cell size. Cells showed lower mobility in vitro, paralleled by reduced protrusions. Constitutive PV down-regulation in PV-overexpressing cells reverted mitochondrial morphology and fractional volume to the state present in control MDCK cells, resulting from increased mitochondrial movement and augmented fusion rates. PV-modulated, bi-directional and reversible mitochondrial dynamics are key to regulation of mitochondrial volume.     

The mesenchymoangioblast,mesodermal precursor for mesenchymal and endothelial cells

Mesenchymoangioblast (MB) is the earliest precursor for endothelial and mesenchymal cells originating from APLNR⁺PDGFRα⁺KDR⁺ mesoderm in human pluripotent stem cell cultures. MBs are identified based on their capacity to form FGF2-dependent compact spheroid colonies in a serum-free semisolid medium. MBs colonies are composed of PDGFRβ⁺CD271⁺EMCN⁺DLK1⁺CD73^? primitive mesenchymal cells which are generated through endothelial/angioblastic intermediates (cores) formed during first 3–4 days of clonogenic cultures. MB-derived primitive mesenchymal cells have potential to differentiate into mesenchymal stromal/stem cells (MSCs), pericytes, and smooth muscle cells. In this review, we summarize the specification and developmental potential of MBs, emphasize features that distinguish MBs from other mesenchymal progenitors described in the literature and discuss the value of these findings for identifying molecular pathways leading to MSC and vasculogenic cell specification, and developing cellular therapies using MB-derived progeny.     

Bafilomycin A1 activates respiration of neuronal cells via uncoupling associated with flickering depolarization of mitochondria

Bafilomycin A1 (Baf) induces an elevation of cytosolic Ca²⁺ and acidification in neuronal cells via inhibition of the V-ATPase. Also, Baf uncouples mitochondria in differentiated PC12 (_dPC12), _dSH-SY5Y cells and cerebellar granule neurons, and markedly elevates their respiration. This respiratory response in _dPC12 is accompanied by morphological changes in the mitochondria and decreases the mitochondrial pH, Ca²⁺ and ΔΨm. The response to Baf is regulated by cytosolic Ca²⁺ fluxes from the endoplasmic reticulum. Inhibition of permeability transition pore opening increases the depolarizing effect of Baf on the ΔΨm. Baf induces stochastic flickering of the ΔΨm with a period of 20 ± 10 s. Under conditions of suppressed ATP production by glycolysis, oxidative phosphorylation impaired by Baf does not provide cells with sufficient ATP levels. Cells treated with Baf become more susceptible to excitation with KCl. Such mitochondrial uncoupling may play a role in a number of (patho)physiological conditions induced by Baf.     

Intra-brain microinjection of human mesenchymal stem cells decreases allodynia in neuropathic mice

Neuropathic pain is a very complex disease, involving several molecular pathways. Current available drugs are usually not acting on the several mechanisms underlying the generation and propagation of pain. We used spared nerve injury model of neuropathic pain to assess the possible use of human mesenchymal stem cells (hMSCs) as anti-neuropathic tool. Human MSCs were transplanted in the mouse lateral cerebral ventricle. Stem cells injection was performed 4 days after sciatic nerve surgery. Neuropathic mice were monitored 7, 10, 14, 17, and 21 days after surgery. hMSCs were able to reduce pain-like behaviors, such as mechanical allodynia and thermal hyperalgesia, once transplanted in cerebral ventricle. Anti-nociceptive effect was detectable from day 10 after surgery (6 days post cell injection). Human MSCs reduced the mRNA levels of the pro-inflammatory interleukin IL-1β mouse gene, as well as the neural β-galactosidase over-activation in prefrontal cortex of SNI mice. Transplanted hMSCs were able to reduce astrocytic and microglial cell activation.     

Megalin mediates plasma membrane to mitochondria cross-talk and regulates mitochondrial metabolism

Mitochondrial intracrines are extracellular signaling proteins, targeted to the mitochondria. The pathway for mitochondrial targeting of mitochondrial intracrines and actions in the mitochondria remains unknown. Megalin/LRP2 mediates the uptake of vitamins and proteins, and is critical for clearance of amyloid-β protein from the brain. Megalin mutations underlie the pathogenesis of Donnai–Barrow and Lowe syndromes, characterized by brain defects and kidney dysfunction; megalin was not previously known to reside in the mitochondria. Here, we show megalin is present in the mitochondria and associates with mitochondrial anti-oxidant proteins SIRT3 and stanniocalcin-1 (STC1). Megalin shuttles extracellularly-applied STC1, angiotensin II and TGF-β to the mitochondria through the retrograde early endosome-to-Golgi transport pathway and Rab32. Megalin knockout in cultured cells impairs glycolytic and respiratory capacities. Thus, megalin is critical for mitochondrial biology; mitochondrial intracrine signaling is a continuum of the retrograde early endosome-to-Golgi-Rab32 pathway and defects in this pathway may underlie disease processes in many systems.     

Biosynthesis and roles of phospholipids in mitochondrial fusion,division and mitophagy

Mitochondria move, fuse and divide in cells. The dynamic behavior of mitochondria is central to the control of their structure and function. Three conserved mitochondrial dynamin-related GTPases (i.e., mitofusin, Opa1 and Drp1 in mammals and Fzo1, Mgm1 and Dnm1 in yeast) mediate mitochondrial fusion and division. In addition to dynamins, recent studies demonstrated that phospholipids in mitochondria also play key roles in mitochondrial dynamics by interacting with dynamin GTPases and by directly changing the biophysical properties of the mitochondrial membranes. Changes in phospholipid composition also promote mitophagy, which is a selective mitochondrial degradation process that is mechanistically coupled to mitochondrial division. In this review, we will discuss the biogenesis and function of mitochondrial phospholipids.     

Binding of estradiol to synaptosomal mitochondria: physiological significance

The subsynaptosomal distribution and specific binding of 17beta-estradiol in vitro to mitochondria isolated from presynaptic nerve endings of female rat brain were examined. 17Beta-estradiol is (i) distributed unequally in synaptosomes and mitochondria posses the highest capacity to bind estradiol with respect to the available amount of the hormone. (ii) Estradiol binds specifically to isolated synaptosomal mitochondria. A Michaelis-Menten plot of specific binding was sigmoidal within a concentration range of 0.1-5 nM of added estradiol, with a saturation plateau at 3 nM. Binding of higher estradiol concentrations demonstrated an exponential Michaelis-Menten plot, indicating non-specific binding to mitochondria. Vmax and Km for the sigmoidal-shape range were estimated as 46 +/- 6 fmol of estradiol/mg of mitochondrial proteins and 0.46 +/- 0.07 nM free estradiol respectively. (iii) Estradiol binding is not affected by the removal of ovaries. The results show that inhibition of Na-dependent Ca2+ efflux from mitochondria by estradiol occurs according to an affinity change of the translocator for Na+, at the same estradiol concentrations that show specific binding to mitochondrial membranes. These data imply that physiological concentrations of estradiol, acting on mitochondrial membrane properties, extragenomically modulate the mitochondrial, and consequently the synaptosomal content of Ca2+, and in that way exert a significant change in nerve cell homeostasis.