Transplantation of adipose-derived mesenchymal stem cells (ASCs) induces tissue regeneration by accelerating the growth of blood vessels and nerve. However, mechanisms by which they accelerate the growth of nerve fibers are only partially understood. We used transplantation of ASCs with subcutaneous matrigel implants (well-known in vivo model of angiogenesis) and model of mice limb reinnervation to check the influence of ASC on nerve growth. Here we show that ASCs stimulate the regeneration of nerves in innervated mice's limbs and induce axon growth in subcutaneous matrigel implants. To investigate the mechanism of this action we analyzed different properties of these cells and showed that they express numerous genes of neurotrophins and extracellular matrix proteins required for the nerve growth and myelination. Induction of neural differentiation of ASCs enhances production of brain-derived neurotrophic factor (BDNF) as well as ability of these cells to induce nerve fiber growth. BDNF neutralizing antibodies abrogated the stimulatory effects of ASCs on the growth of nerve sprouts. These data suggest that ASCs induce nerve repair and growth via BDNF production. This stimulatory effect can be further enhanced by culturing the cells in neural differentiation medium prior to transplantation.

Adipose-derived stromal cells (ASCs) are suggested to be potent candidates for cell therapy of ischemic conditions due to their ability to stimulate blood vessel growth. ASCs produce many angiogenic and anti-apoptotic growth factors, and their secretion is significantly enhanced by hypoxia. Utilizing a Matrigel implant model, we showed that hypoxia-treated ASCs stimulated angiogenesis as well as maturation of the newly formed blood vessels in vivo. To elucidate mechanisms of ASC angiogenic action, we used a co-culture model of ASCs with cells isolated from early postnatal hearts (cardiomyocyte fraction, CMF). CMF contained mature cardiomyocytes, endothelial cells, and progenitor cells. On the second day of culture CMF cells formed spontaneously beating colonies with CD31+ capillary-like structures outgrowing from those cell aggregates. However, these vessel-like structures were not stable, and disassembled within next 5 days. Co-culturing of CMF with ASCs resulted in the formation of stable and branched CD31+ vessel-like structures. Using immunomagnetic depletion of CMF from vascular cells as well as incubation of CMF with mitomycin C-treated ASCs, we showed that in co-culture ASCs enhance blood vessel growth not only by production of paracrine-acting factors but also by promoting the endothelial differentiation of cardiac progenitor cells. All these mechanisms of actions could be beneficial for the stimulation of angiogenesis in ischemic tissues by ASCs administration.

Urokinase-type plasminogen activator (uPA) participates in diverse (patho)physiological processes through intracellular signaling events that affect cell adhesion, migration, and proliferation, although the mechanisms by which these occur are only partially understood. Here we report that upon cell binding and internalization, single-chain uPA (scuPA) translocates to the nucleus within minutes. Nuclear translocation does not involve proteolytic activation or degradation of scuPA. Neither the urokinase receptor (uPAR) nor the low-density lipoprotein-related receptor (LRP) is required for nuclear targeting. Rather, translocation involves the binding of scuPA to the nucleocytoplasmic shuttle protein nucleolin through a region containing the kringle domain. RNA interference and mutational analysis demonstrate that nucleolin is required for the nuclear transport of scuPA. Furthermore, nucleolin is required for the induction smooth muscle ␣-actin (␣-SMA) by scuPA. These data reveal a novel pathway by which uPA is rapidly translocated to the nucleus where it might participate in regulating gene expression. (Blood. 2008;112: 100-110) IntroductionUrokinase-type plasminogen activator (uPA) is a multifunctional protein that has been implicated in several physiological and pathological processes, including cell proliferation and migration during angiogenesis, tissue regeneration, inflammatory responses, and tumor growth/metastases. These complex processes all involve intracellular signal transduction and regulation of gene transcription in addition to proteolysis (see Alfano et al 1 for review). uPA is secreted as a single-chain protein (scuPA) that consists of an N-terminal EGF-like domain (GFD), a kringle domain (KD), and a serine protease domain. Binding of uPA to its high-affinity receptor CD87 (uPAR) is mediated by the GFD. 2 Plasmin converts scuPA into a proteolytically active 2-chain enzyme (tcuPA) 3 that is rapidly inhibited primarily by plasminogen activator inhibitor-1 (PAI-1). tcuPA-PAI-1 complexes are internalized with the aid of lipoprotein receptor-related protein (LRP) 4 by clathrin-mediated endocytosis. The tcuPA-PAl-1 complexes traffic to lysosomes and are degraded, while unoccupied uPAR and LRP recycle back to the cell surface. 5 uPA-induced signal transduction occurs via uPAR-dependent and uPAR-independent pathways (reviewed in Alfano et al 1 ; Kjoller 6 ; Blasi and Carmeliet 7 ). Among the latter, we have shown that cleavage of scuPA by plasmin releases the GFD fragment, generating a form of uPA unable to bind to uPAR, 8 but that stimulates migration of smooth muscle cells (SMCs). 9 Signal transduction by this scuPA fragment may be mediated in part by LRP 10 and certain integrins. 11 However, there is limited information as to the mechanism by which uPA modifies gene transcription, [12][13][14][15] and our previous studies have provided reason to hypothesize that cells express additional uPA-binding proteins that possess distinct signal-transducing activities involved in cell contractility, migration, an...

BackgroundChronic heart failure (CHF) is increasing in prevalence. Patients with CHF usually have co-morbid conditions, but these have been subjected to little research and consequently there is a paucity of guidance on how to manage them. Obesity and diabetes mellitus are common antecedents of CHF and often complicate management and influence outcome. Cachexia is an ominous and often missed sign in patients with CHF.MethodsThis manuscript describes the rationale and the design of Studies Investigating Co-morbidities Aggravating Heart Failure (SICA-HF), a prospective, multicentre, multinational, longitudinal, pathophysiological evaluation study, which is being conducted in 11 centres across six countries in the European Union and in Russia. We aim to recruit >1,600 patients with CHF due to various common aetiologies, irrespective of left ventricular ejection fraction, and with or without co-morbidities at study entry. In addition, >300 patients with type 2 diabetes mellitus without CHF and >150 healthy subjects will serve as control groups. Participants will be systematically investigated at annual intervals for up to 48 months. Additional investigations focusing on cellular and subcellular mechanisms, adipose and skeletal muscle tissue, and in endothelial progenitor cells will be performed in selected subgroups.ConclusionsSICA-HF will provide insights into common co-morbidities in CHF with a specific emphasis on diabetes mellitus and body mass. This will provide a more thorough pathophysiological understanding of the complexity of CHF that will help develop therapies tailored to manage specific co-morbidities.

Objective-We showed previously that increased urokinase plasminogen activator (uPA) expression contributes to vascular smooth muscle cell (VSMC) proliferation and neointima formation after injury. Proliferation of cultured rat aortic VSMCs induced by uPA was inhibited by the antioxidant ebselen. Because increases in VSMC reactive oxygen species (ROS) contribute to VSMC proliferation, we hypothesized that uPA increases ROS generation by regulating expression or activity of cellular oxidases. Methods and Results-uPA stimulated ROS production to levels equivalent to angiotensin II as measured by electron spin resonance and fluorescent redox indicators (dichlorofluorescein diacetate, lucigenin, and hydroethidine). The increase in ROS was biphasic, with the first peak at 30 minutes and the second peak at 4 hours. uPA increased expression of the NAD(P)H oxidases Nox1 and Nox4 as measured by RT-PCR and Western blot analysis. Knockdown of Nox1 and Nox4 expression with small interfering RNA showed that both isoforms (Nox1ϾNox4) contributed significantly to uPA-stimulated ROS production and VSMC proliferation. Transfection of VSMCs with uPA cDNA to increase endogenous uPA expression enhanced ROS production dramatically, suggesting that autocrine uPA production may be an important mechanism for uPA-mediated VSMC events. Conclusion-These data show that uPA is an autocrine VSMC growth factor that increases ROS generated by both Nox1 and Nox4 oxidases. Key Words: urokinase Ⅲ superoxide Ⅲ VSMC proliferation Ⅲ arterial remodeling P lasminogen activators, their inhibitors, and receptors are the main components of the fibrinolytic (or plasminogen/ plasmin) system that, together with the blood coagulation system, determine the balance between the formation and dissolution of blood clots. 1 In addition, much evidence suggests that the fibrinolytic system also participates in vascular remodeling processes such as atherosclerosis and postangioplasty restenosis. [2][3][4][5][6] The predominant processes that contribute to vessel remodeling in cardiovascular diseases are proliferation and migration of vascular smooth muscle cells (VSMCs), extracellular matrix deposition, adhesion of inflammatory cells, invasion into the vessel wall, and proliferation. These processes are regulated by growth factors and the components of the fibrinolytic or plasminogen system. 7,8 The plasminogen system is composed of an inactive proenzyme, plasminogen, that is converted to active plasmin by 2 physiological plasminogen activators: tissue-type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA). 1,9 VSMCs use proteinases to degrade the extracellular matrix that encages them, releasing them to migrate into the wound. 10 Plasmin may trigger this process because it can directly degrade fibrin and matrix and also activate other matrix-degrading proteinases, including the metalloproteinases. Plasmin has been presumed to play a role in tissue remodeling via proteolysis of extracellular matrix components and activation of growth factors. ...