Simvastatin Modulates Mesenchymal Stromal Cell Proliferation and Gene Expression

<div><p>Statins are widely used hypocholesterolemic drugs that block the mevalonate pathway, responsible for the biosysnthesis of cholesterol. However, statins also have pleiotropic effects that interfere with several signaling pathways. Mesenchymal stromal cells (MSC) are a heterogeneous mixture of cells that can be isolated from a variety of tissues and are identified by the expression of a panel of surface markers and by their ability to differentiate <i>in vitro</i> into osteocytes, adipocytes and chondrocytes. MSC were isolated from amniotic membranes and bone marrows and characterized based on ISCT (International Society for Cell Therapy) minimal criteria. Simvastatin-treated cells and controls were directly assayed by CFSE (Carboxyfluorescein diacetate succinimidyl ester) staining to assess their cell proliferation and their RNA was used for microarray analyses and quantitative PCR (qPCR). These MSC were also evaluated for their ability to inhibit PBMC (peripheral blood mononuclear cells) proliferation. We show here that simvastatin negatively modulates MSC proliferation in a dose-dependent way and regulates the expression of proliferation-related genes. Importantly, we observed that simvastatin increased the percentage of a subset of smaller MSC, which also were actively proliferating. The association of MSC decreased size with increased pluripotency and the accumulating evidence that statins may prevent cellular senescence led us to hypothesize that simvastatin induces a smaller subpopulation that may have increased ability to maintain the entire pool of MSC and also to protect them from cellular senescence induced by long-term cultures/passages <i>in vitro</i>. These results may be important to better understand the pleiotropic effects of statins and its effects on the biology of cells with regenerative potential.</p></div

MSC subpopulations by size (FSC) and complexity (SSC).

<p>The different subsets are surrounded by a blue line drawn around them and the values correspond to the percentage of each in this example. S_5uM, MSC treated with simvastatin in the concentration of 5μM. S+M, MSC treated with 5μM of simvastatin and 100μM of activated L-Mevalonate (M).</p

CFSE proliferation assays of MSC.

<p>The values correspond to the median percentage of viable, CFSE+ cells. S_1uM: MSC treated with simvastatin in the concentration of 1μM. S_5uM, MSC treated with simvastatin in the concentration of 5μM. S+M, MSC treated with 5μM of simvastatin and 100μM of activated L-Mevalonate (M). S+GGPP, MSC treated with 5μM of simvastatin and 5μM of GGPP (20-carbon geranylgeranyl pyrophosphate).</p

Analysis of MSC proliferation for the quadrant of small (FSC^lo) and low complexity (SSC^lo) cells.

<p>S_1uM: MSC treated with simvastatin in the concentration of 1μM. S_5uM, MSC treated with simvastatin in the concentration of 5μM. S+M, MSC treated with 5μM of simvastatin and 100μM of activated L-Mevalonate (M). S+GGPP, MSC treated with 5μM of simvastatin and 5μM of GGPP (20-carbon geranylgeranyl pyrophosphate). A) gating strategy to remove debril and dead cells; B) FSC and SSC quadrants definition; C) Percentages of cells with FSC and SSC< 200; D) Percentages of cells with FSC and SSC> 200.</p

Median values of CFSE+ PBMC to assess the ability of simvastatin-treated MSC to inhibit PBMC proliferation.

<p>PBMC alone correspond to the proliferation rate of PBMC alone. Control refers to co-cultures of PBMC with MSC samples exposed only to the vehicle (absolute ethanol). S_1uM: MSC treated with simvastatin in the concentration of 1μM. S_5uM, MSC treated with simvastatin in the concentration of 5μM. S+M, MSC treated with 5μM of simvastatin and 100μM of activated L-Mevalonate (M).</p

Analysis of MSC proliferation for A) the quadrant of small (FSC^lo) and proliferative (CFSE^hi) MSC and for B) quadrant of large (FSC^hi) and non-proliferative (CFSE^lo) MSC.

<p>S_1uM: MSC treated with simvastatin in the concentration of 1μM. S_5uM, MSC treated with simvastatin in the concentration of 5μM. S+M, MSC treated with 5μM of simvastatin and 100μM of activated L-Mevalonate (M). S+GGPP, MSC treated with 5μM of simvastatin and 5μM of GGPP (20-carbon geranylgeranyl pyrophosphate).</p

Validation of microarray experiments by quantitative real time PCR (qRT-PCR) for selected genes.

<p>The graph shows the median of 2^-dCt values, agains the geometric mean of the dCt of two reference genes. Control refers to MSC exposed only to the vehicle (absolute ethanol). S_1Um and S_5uM: MSC treated with simvastatin in the concentration of 1μM and 5μM, respectively. S+FPP and S+GGPP, MSC treated with 5μM of simvastatin and 5μM of FPP (15-carbon farnesylpyrophosphate) and 5μM of GGPP (20-carbon geranylgeranyl pyrophosphate), respectively. S+M, MSC treated with 5μM of simvastatin and 100μM of activated L-Mevalonate (M).</p