Grigorios Tsaknakis

Tsc1 (hamartin) confers neuroprotection against ischemia by inducing autophagy

Previous attempts to identify neuroprotective targets by studying the ischemic cascade and devising ways to suppress it have failed to translate to efficacious therapies for acute ischemic stroke. We hypothesized that studying the molecular determinants of endogenous neuroprotection in two well-established paradigms, the resistance of CA3 hippocampal neurons to global ischemia and the tolerance conferred by ischemic preconditioning (IPC), would reveal new neuroprotective targets. We found that the product of the tuberous sclerosis complex 1 gene (TSC1), hamartin, is selectively induced by ischemia in hippocampal CA3 neurons. In CA1 neurons, hamartin was unaffected by ischemia but was upregulated by IPC preceding ischemia, which protects the otherwise vulnerable CA1 cells. Suppression of hamartin expression with TSC1 shRNA viral vectors both in vitro and in vivo increased the vulnerability of neurons to cell death following oxygen glucose deprivation (OGD) and ischemia. In vivo, suppression of TSC1 expression increased locomotor activity and decreased habituation in a hippocampal-dependent task. Overexpression of hamartin increased resistance to OGD by inducing productive autophagy through an mTORC1-dependent mechanism.

Human endothelial stem/progenitor cells, angiogenic factors and vascular repair

Neovascularization or new blood vessel formation is of utmost importance not only for tissue and organ development and for tissue repair and regeneration, but also for pathological processes, such as tumour development. Despite this, the endothelial lineage, its origin, and the regulation of endothelial development and function either intrinsically from stem cells or extrinsically by proangiogenic supporting cells and other elements within local and specific microenvironmental niches are still not fully understood. There can be no doubt that for most tissues and organs, revascularization represents the holy grail for tissue repair, with autologous endothelial stem/progenitor cells, their proangiogenic counterparts and the products of these cells all being attractive targets for therapeutic intervention. Historically, a great deal of controversy has surrounded the identification and origin of cells and factors that contribute to revascularization, the use of such cells or their products as biomarkers to predict and monitor tissue damage and repair or tumour progression and therapeutic responses, and indeed their efficacy in revascularizing and repairing damaged tissues. Here, we will review the role of endothelial progenitor cells and of supporting proangiogenic cells and their products, principally in humans, as diagnostic and therapeutic agents for wound repair and tissue regeneration.

Spindle Shaped Human Mesenchymal Stem/Stromal Cells from Amniotic Fluid Promote Neovascularization

Human amniotic fluid obtained at amniocentesis, when cultured, generates at least two morphologically distinct mesenchymal stem/stromal cell (MSC) subsets. Of these, the spindle shaped amniotic fluid MSCs (SS-AF-MSCs) contain multipotent cells with enhanced adipogenic, osteogenic and chondrogenic capacity. Here, we demonstrate, for the first time, the capacity of these SS-AF-MSCs to support neovascularization by umbilical cord blood (UCB) endothelial colony forming cell (ECFC) derived cells in both in vitro and in vivo models. Interestingly, although the kinetics of vascular tubule formation in vitro were similar when the supporting SS-AF-MSCs were compared with the best vasculogenic supportive batches of bone marrow MSCs (BMSCs) or human dermal fibroblasts (hDFs), SS-AF-MSCs supported vascular tubule formation in vivo more effectively than BMSCs. In NOD/SCID mice, the human vessels inosculated with murine vessels demonstrating their functionality. Proteome profiler array analyses revealed both common and distinct secretion profiles of angiogenic factors by the SS-AF-MSCs as opposed to the hDFs and BMSCs. Thus, SS-AF-MSCs, which are considered to be less mature developmentally than adult BMSCs, and intermediate between adult and embryonic stem cells in their potentiality, have the additional and very interesting potential of supporting increased neovascularisation, further enhancing their promise as vehicles for tissue repair and regeneration.

The impact of proliferative potential of umbilical cord-derived endothelial progenitor cells and hypoxia on vascular tubule formation in vitro.

Revascularization of the damaged tissue is pivotal to tissue repair. Here, by bringing together two in vitro model systems, we have been able to examine (1) the ability of human umbilical vein endothelial cells (HUVEC) containing a complete hierarchy of endothelial progenitors derived from the human umbilical cord to generate vascular tubules within a human stromal niche in vitro and (2) the effects of exposure to low oxygen tensions on endothelial progenitor cell proliferation and tubule formation in vitro. Our results demonstrate that high proliferative potential endothelial colony forming cells (HPP-ECFC) from cultured HUVEC preferentially contribute to vascular tubule formation in vitro and that these progenitor cells are concentrated in the CD34(lo/-) fraction. HUVEC were initially resistant when exposed to hypoxia (1.5% O(2)) for short periods (1-2 days), but sustained chronic hypoxia (4-14 days) inhibited their ability to proliferate. This was reflected by a loss in their ability to form tubules in cocultures of human dermal fibroblasts (hDFs). In contrast, an acute exposure to low oxygen tensions (1.5% O(2) for 24 h) followed by reoxygenation did not adversely affect the capacity of these cells to both proliferate and form vascular tubules in vitro.These studies therefore provide a model system to study the influences of the microenvironmental niche and modification of this niche on vascular tubule formation in vitro from HPP-ECFC.

A novel function for the haemopoietic supportive murine bone marrow MS‐5 mesenchymal stromal cell line in promoting human vasculogenesis and angiogenesis

The bone marrow contains specific microenvironmental stem cell niches that maintain haemopoiesis. CXCL12‐expressing mesenchymal stromal cells are closely associated with the bone marrow sinusoidal endothelia, forming key elements of the haemopoietic stem cell niche, yet their ability to regulate endothelial function is not clearly defined. Given that the murine nestin+ cell line, MS‐5, provides a clonal surrogate bone marrow stromal niche capable of regulating both murine and human primitive haemopoietic stem/progenitor cell (HSC/HPC) fate in vitro, we hypothesized that MS‐5 cells might also support new blood vessel formation and function. Here, for the first time, we demonstrate that this is indeed the case. Using proteome arrays, we identified HSC/HPC active angiogenic factors that are preferentially secreted by haemopoietic supportive nestin+MS‐5 cells, including CXCL12 (SDF‐1), NOV (CCN3), HGF, Angiopoietin‐1 and CCL2 (MCP‐1). Concentrating on CXCL12, we confirmed its presence in MS‐5 conditioned media and demonstrated that its antagonist in receptor binding, AMD‐3100, which mobilizes HSC/HPCs and endothelial progenitors from bone marrow, could significantly reduce MS‐5 mediated human vasculogenesis in vitro, principally by regulating human endothelial cell migration. Thus, the clonal nestin+MS‐5 murine bone marrow stromal cell line not only promotes human haemopoiesis but also induces human vasculogenesis, with CXCL12 playing important roles in both processes.

Microvessel networks in pre-formed in artificial clinical grade dermal substitutes in vitro using cells from haematopoietic tissues

Forming a microcirculation is critical for vascularisation of artificial skin substitutes. One strategy to improve speed of grafting is to pre-form microvascular networks in the substitute before applying to a wound. For clinical application, this requires sufficient functional endothelial cell numbers. In vitro endothelial colony forming cells (ECFCs) derived cells were expanded from cord and adult blood donations and co-cultured with human dermal fibroblasts or bone marrow mesenchymal stem/stromal cells to form microvascular networks in the presence or absence of dermal substitutes which are in clinical use. The number of endothelial cells generated ranged from 1.03×10(9) to 2.18×10(11) from 10 adult blood donations and 1×10(12) to 1.76×10(13) from 6 cord blood units after 50 days in culture. Two adult donations failed to generate ECFCs. Both cord and adult blood cells formed 2D microvascular networks in vitro, although there was a significant difference in the functional capacity of adult and cord blood ECFCs. While co-culture of the latter within dermal substitutes Matriderm or Integra demonstrated the formation of 3D microvascular networks penetrating 100μm, enhanced expansion, while maintaining functional capacity, of adult blood cells is required for fully pre-vascularising the clinical grade acellular dermal substitutes used here prior to applying these to burns.

Effects of obstetric factors and storage temperatures on the yield of endothelial colony forming cells from umbilical cord blood

As umbilical cord blood (UCB) is a rich source of endothelial colony-forming cells (ECFC), our aim was twofold: (1) to examine potential obstetric selection criteria for achieving the highest ECFC yields from UCB units, and (2) to determine whether transient storage temperatures of fresh UCB and cryopreservation of UCB units affected ECFC yield and function. ECFC quality was assessed before and after cryopreservation by their clonogenic proliferative potential. Of the 20 factors examined, placental weight was the only statistically significant obstetric factor that predicted ECFC frequency in UCB. Studies on the effects of storage revealed that transient storage of fresh UCB at 4°C reduced ECFC yield compared with storage at 22°C, while cryopreservation of UCB MNCs significantly reduced ECFC recoveries. To our knowledge, this is the first demonstration that placental weight and temperature of storage prior to processing or culture have significant effects on ECFC frequency in UCB. Our studies further support the evidence that cryopreservation of UCB MNCs compromises ECFC recovery.

The Murine Ortholog of the SHP-2 Binding Molecule, PZR Accelerates Cell Migration on Fibronectin and Is Expressed in Early Embryo Formation

The human P zero‐related protein (hPZR) has a unique function in regulating cell migration. This activity is dependent on both its cytoplasmic immunoreceptor tyrosine inhibitory motif (ITIM) and its interaction with the tyrosine protein phosphatase, src homology phosphatase‐2 (SHP‐2). Here, using in silico and cDNA cloning approaches, we identify the murine ITIM‐containing hPZR ortholog, mPZR, together with its ITIM‐less isoform, mPZRb. We demonstrate that, like hPZR, these type 1 integral murine transmembrane isoforms are derived by differential splicing from a single gene transcription unit on mouse chromosome 1, and differ only in the sequence of their cytoplasmic domains. Importantly, mPZR mimicks hPZR functionally by accelerating SHP‐2‐mediated cell migration on fibronectin. Interestingly, we further demonstrate that although neither mPZR nor mPZRb is expressed in murine pluripotent embryonic stem cells, they first appear at approximately day 3 of blastocyst formation in vivo and of embryoid body formation in vitro. These studies thus provide the basis for defining the function of the mPZR isoforms in vivo, particularly with respect to their roles in regulating SHP‐2‐dependent cell migration during development. J. Cell. Biochem. 102: 955–969, 2007.

miR-193a-3p interaction with HMGB1 downregulates human endothelial cell proliferation and migration

Circulating endothelial colony forming cells (ECFCs) contribute to vascular repair where they are a target for therapy. Since ECFC proliferative potential is increased in cord versus peripheral blood and to define regulatory factors controlling this proliferation, we compared the miRNA profiles of cord blood and peripheral blood ECFC-derived cells. Of the top 25 differentially regulated miRNAs selected, 22 were more highly expressed in peripheral blood ECFC-derived cells. After validating candidate miRNAs by q-RT-PCR, we selected miR-193a-3p for further investigation. The miR-193a-3p mimic reduced cord blood ECFC-derived cell proliferation, migration and vascular tubule formation, while the miR-193a-3p inhibitor significantly enhanced these parameters in peripheral blood ECFC-derived cells. Using in silico miRNA target database analyses combined with proteome arrays and luciferase reporter assays of miR-193a-3p mimic treated cord blood ECFC-derived cells, we identified 2 novel miR-193a-3p targets, the high mobility group box-1 (HMGB1) and the hypoxia upregulated-1 (HYOU1) gene products. HMGB1 silencing in cord blood ECFC-derived cells confirmed its role in regulating vascular function. Thus, we show, for the first time, that miR-193a-3p negatively regulates human ECFC vasculo/angiogenesis and propose that antagonising miR-193a-3p in less proliferative and less angiogenic ECFC-derived cells will enhance their vasculo/angiogenic function.

Stem Cells, Hypoxia and Hypoxia-Inducible Factors

Oxygen is a critical environmental factor that regulates the fate of stem cells. In this review, our aims are twofold: (i) to consider the contribution of oxygen tension to the environmental niches in which stem cells and their progeny find themselves and which have a role in determining their fate, and (ii) to define the regulatory networks that control the response of stem/progenitor cells to hypoxia, particularly those that affect early embryonic stem cell specification and hematopoietic stem cell development and function.