Jeffrey L. Spees - US grants

DESCRIPTION (provided by applicant): There is increasing interest in exploring the potential therapeutic uses of adult stem cells from bone marrow. Currently, however, it is not understood which stem cell populations or cellular phenotypes are best suited to effectively treat a variety of diseases that may be amenable to such a therapeutic approach. There is very little fundamental information on the basic biology of adult bone marrow stem cells and whether or not they will provide a source of cells that can repair damaged tissues and ameliorate a disease process. Thus, we have proposed a series of experiments to test the central hypothesis that purified sub-populations of adult rat bone marrow-derived stem cells are effective in repairing injured cardiac and pulmonary tissues. Three specific aims have been developed to directly test the hypothesis: Specific Aim: 1. To establish the morphologic and molecular profiles of purified adult stem cell sub-populations from the bone marrow of male transgenic rats that ubiquitously express green fluorescent protein (GFP). 2. To determine the differentiation potential of the stem cell sub-populations in ex vivo co-culture assays we have recently developed with normal and injured primary cells from cardiac and pulmonary tissues. 3. To determine the engraftment and differentiation potential of the stem cell sub-populations in a rat model of monocrotaline-induced pulmonary hypertension.

[unreadable] DESCRIPTION (provided by applicant): The adult bone marrow non-hematopoietic stem cells known as mesenchymal stem cells (MSCs) promote functional cardiac recovery in animals with myocardial infarction (MI). In several countries these cells have been administered to patients with MI with reported beneficial effects. Despite the efficacy of MSC administration, most studies have shown that few of the cells engraft long-term and that only a portion of the surviving cells appear to differentiate to a mature cardiac phenotype. In animals injection of concentrated conditioned medium into cardiac muscle appears to have effects similar to those of direct injection of cells, implying that secreted factors from the cells comprise the principle basis for the benefits. Our preliminary data demonstrate that non-hematopoietic human bone marrow stem cells can exert cardiac protective and reparative effects when delivered intravenously (IV) to animals with MI. Furthermore, we show that serum-free medium conditioned by mixed MSCs or by standardized MSCs isolated by magnetic sorting for the p75 low affinity nerve growth factor receptor (p75LNGFR; p75MSCs) can support the growth and survival of adult cardiac stem/progenitor cells through activation of the transcription factor STAT3. Because MSCs are prepared from a heterogeneous mixture of adherent cells, it is desirable to have a standardized isolation procedure for a population of progenitor cells that predictively imparts cardiac protection and/or repair. We have recently isolated sub-populations of non-hematopoietic stem cells from the total mononuclear cells of bone marrow by specific cell surface epitopes. They may be particularly well-suited for cardiac preservation based on their expression profiles of secreted proteins. Comparing the effectiveness of one of these sub-populations (p75MSCs) to mixed adherent MSCs, we will determine whether intravenous or intramuscular administration of non-autologous stem cells can be used to provide paracrine-based cardiac cell therapy. We will identify the factors secreted by the stem cells that are effective and determine whether the mechanism of action is sparing of existing cardiomyocytes by inhibition of apoptosis or necrosis or augmenting the proliferation and survival of endogenous cardiac stem cells. [unreadable] [unreadable] SPECIFIC AIMS [unreadable] [unreadable] 1. To identify a cardioprotective sub-population of human bone marrow stem cells and determine the growth conditions and timing of administration necessary to rescue cardiac function after MI in immunodeficient mice. [unreadable] [unreadable] 2. To determine the ability of strain-mismatched murine bone marrow stem cells delivered intravenously or intramuscularly to promote cardiac protection or recovery after MI in immunocompetent mice. [unreadable] [unreadable] 3. To identify the specific factors secreted by the stem cells that preserve cardiac function. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Reactive astrogliosis and the subsequent formation of a glial scar are robust phenomena that occur following diverse CNS injuries. Surprisingly, the molecular signals that control the proliferation of reactive astrocytes or their functions in vivo are poorly understood. Defining the molecular control of reactive astrocyte proliferation and function may lead to therapeutic strategies that modify specific signals in reactive astrocytes to preserve tissue or improve recovery after CNS injury. We demonstrate that intra-arterial infusion of the gamma secretase (GS) inhibitor DBZ (Dibenzazepine) after stroke significantly reduced the proliferation of reactive astrocytes in the peri-infarct area of the cortex, significantly reduced the expression of glial fibrillary acidic protein (GFAP, a marker of activated hypertrophic astrocytes), and significantly increased stroke infarct volumes. The absence of reactive astrocytes after stroke and DBZ treatment correlated with a significant increase in the number of CD45-positive inflammatory cells that invaded the stroke penumbra. Similarly, stereotaxic injection of DBZ directly into the cortex reduced the numbers of proliferating reactive astrocytes surrounding the brain stab injury (needle track) compared with vehicle-injected controls. Reactive astrocytes surrounding the brain stab injury that remained after DBZ injection possessed an altered morphology with a significant reduction in average number of processes, number of branch points, and number of branch ends. Immunohistochemistry with antisera specific to GS cleavage products demonstrated nuclear localization of NICD1 (Notch1) and AICD in reactive astrocytes after cortical injury. DBZ blocks the catalytic activity of Presenilin 1, a component of GS. Experiments designed to specifically delete Notch1 and APP from reactive astrocytes prior to stroke using conditional knockout mice demonstrated that both regulate cortical reactive astrocytes in the peri-infarct area after stroke. Collectively our results indicate that Presenilin 1, Notch1, and APP regulate reactive astrocytes after stroke. Specific Aims: 1. To determine if Presenilin 1 acts as a global regulator of reactive astrogliosis after stroke. 2. To determine whether Notch1 or APP signaling controls the proliferation, morphology, and/or anti-inflammatory functions of reactive astrocytes after stroke. 3. To determine whether Presenilin 1, Notch1, or APP expression in reactive astrocytes is necessary for repair of the blood brain barrier after stroke.

? DESCRIPTION: The Central Hypothesis is treatment regimes that protect vascular integrity early after reperfusion will reduce infarct expansion, final infarct size, and adverse cardiac remodeling in hearts with myocardial infarction. A no re-flow phenomenon is commonly observed in experimental animals with myocardium subjected to ischemia followed by reperfusion and in patients with acute myocardial infarction (MI). No re-flow refers to compromised distal myocardial perfusion despite restoration of patency in proximal macroscopic vessels. The extent of no re-flow is a determinant of infarct expansion. No re-flow may result from destruction of microscopic vessels, which we have termed vascular rhexis, or from other factors such as microemboli, inflammation, release of toxic cellular metabolites, and oxidative stress that cause endothelial cell dysfunction and induce microvascular leaks. This proposal is based on the concept that treatments designed to improve blood supply by preventing vascular rhexis associated with ischemia and reperfusion will reduce no re-flow and diminish infarct size, adverse ventricular remodeling and dysfunction after MI. SPECIFIC AIMS: 1. Determine whether treatment with HGF/IgG complexes at the time of reperfusion can promote vascular integrity and reduce infarct expansion in a large animal model of MI. 2. Identify signaling pathway(s) activated by HGF/IgG complexes that confer enhanced vaso- protection after MI. 3. Determine whether sustained treatment with HGF/IgG complexes after MI provides long- term improvement in cardiac function by increasing cardiac perfusion and reducing infarct expansion, final infarct size, and/or adverse ventricular remodeling.