Rosendo Estrada

Secretome from mesenchymal stem cells induces angiogenesis via Cyr61.

It is well known that bone marrow‐derived mesenchymal stem cells (MSCs) are involved in wound healing and regeneration responses. In this study, we globally profiled the proteome of MSCs to investigate critical factor(s) that may promote wound healing. Cysteine‐rich protein 61 (Cyr61) was found to be abundantly present in MSCs. The presence of Cyr61 was confirmed by immunofluorescence staining and immunoblot analysis. Moreover, we showed that Cyr61 is present in the culture medium (secretome) of MSCs. The secretome of MSCs stimulates angiogenic response in vitro, and neovascularization in vivo. Depletion of Cyr61 completely abrogates the angiogenic‐inducing capability of the MSC secretome. Importantly, addition of recombinant Cyr61 polypeptides restores the angiogenic activity of Cyr61‐depleted secretome. Collectively, these data demonstrate that Cyr61 polypeptide in MSC secretome contributes to the angiogenesis‐promoting activity, a key event needed for regeneration and repair of injured tissues. J. Cell. Physiol. 219: 563–571, 2009.

Balance of S1P1 and S1P2 signaling regulates peripheral microvascular permeability in rat cremaster muscle vasculature

Sphingosine-1-phosphate (S1P) regulates various molecular and cellular events in cultured endothelial cells, such as cytoskeletal restructuring, cell-extracellular matrix interactions, and intercellular junction interactions. We utilized the venular leakage model of the cremaster muscle vascular bed in Sprague-Dawley rats to investigate the role of S1P signaling in regulation of microvascular permeability. S1P signaling is mediated by the S1P family of G protein-coupled receptors (S1P(1-5) receptors). S1P(1) and S1P(2) receptors, which transduce stimulatory and inhibitory signaling, respectively, are expressed in the endothelium of the cremaster muscle vasculature. S1P administration alone via the carotid artery was unable to protect against histamine-induced venular leakage of the cremaster muscle vascular bed in Sprague-Dawley rats. However, activation of S1P(1)-mediated signaling by SEW2871 and FTY720, two agonists of S1P(1), significantly inhibited histamine-induced microvascular leakage. Treatment with VPC 23019 to antagonize S1P(1)-regulated signaling greatly potentiated histamine-induced venular leakage. After inhibition of S1P(2) signaling by JTE-013, a specific antagonist of S1P(2), S1P was able to protect microvascular permeability in vivo. Moreover, endothelial tight junctions and barrier function were regulated by S1P(1)- and S1P(2)-mediated signaling in a concerted manner in cultured endothelial cells. These data suggest that the balance between S1P(1) and S1P(2) signaling regulates the homeostasis of microvascular permeability in the peripheral circulation and, thus, may affect total peripheral vascular resistance.

Glycero- versus sphingo-phospholipids: correlations with human and non-human mammalian lens growth

The human lens differs from other mammalian lenses in its very slow growth and unusual phospholipid composition of its cell membranes. Dihydrosphingomyelins (DHSMs) make up about half of all phospholipids in adult human fiber membranes. In all other membranes, sphingomyelins(SMs) with a trans double bond in their backbone, are prevalent. In our quest to understand the biological implications of such elevated DHSM levels, we analyzed membranes from various regions of human, elephant, giraffe, polar bear, pig and cow lenses. The levels of DHSMs were minor in non-human lens membranes. A strong correlation was observed between growth rate and relative contents of phosphatidylcholines(PCs) in epithelia and outer cortical fibers. Sphingomyelins became increasingly predominant in differentiated fibers and this increase was age dependent. Indeed, nuclear fiber membranes of aged non-human mammals were composed, almost exclusively, of (SMs). Although human lens membranes followed comparable compositional trends, the magnitude of the changes was much smaller. We postulate that the high relative contents of DHSMs provide a biochemically inert matrix in which only small amounts of PCs and SMs and their metabolites, known to promote and arrest growth, respectively, are present. This compositional difference is proposed to contribute to the slow multiplication and elongation of human lens cells.

Microfluidic cardiac cell culture model (μCCCM).

Physiological heart development and cardiac function rely on the response of cardiac cells to mechanical stress during hemodynamic loading and unloading. These stresses, especially if sustained, can induce changes in cell structure, contractile function, and gene expression. Current cell culture techniques commonly fail to adequately replicate physical loading observed in the native heart. Therefore, there is a need for physiologically relevant in vitro models that recreate mechanical loading conditions seen in both normal and pathological conditions. To fulfill this need, we have developed a microfluidic cardiac cell culture model (μCCCM) that for the first time allows in vitro hemodynamic stimulation of cardiomyocytes by directly coupling cell structure and function with fluid induced loading. Cells are cultured in a small (1 cm diameter) cell culture chamber on a thin flexible silicone membrane. Integrating the cell culture chamber with a pump, collapsible pulsatile valve and an adjustable resistance element (hemostatic valve) in series allow replication of various loading conditions experienced in the heart. This paper details the design, modeling, fabrication and characterization of fluid flow, pressure and stretch generated at various frequencies to mimic hemodynamic conditions associated with the normal and failing heart. Proof-of-concept studies demonstrate successful culture of an embryonic cardiomyoblast line (H9c2 cells) and establishment of an in vivo like phenotype within this system.

PEDF from mouse mesenchymal stem cell secretome attracts fibroblasts

Conditioned medium (secretome) derived from an enriched stem cell culture stimulates chemotaxis of human fibroblasts. These cells are classified as multipotent murine mesenchymal stromal cells (mMSC) by immunochemical analysis of marker proteins. Proteomic analysis of mMSC secretome identifies nineteen secreted proteins, including extracellular matrix structural proteins, collagen processing enzymes, pigment epithelium‐derived factor (PEDF) and cystatin C. Immunodepletion and reconstitution experiments show that PEDF is the predominant fibroblast chemoattractant in the conditioned medium, and immunofluorescence microscopy shows strong staining for PEDF in the cytoplasm, at the cell surface, and in intercellular space between mMSCs. This stimulatory effect of PEDF on fibroblast chemotaxis is in contrast to the PEDF‐mediated inhibition of endothelial cell migration, reported previously. These differential functional effects of PEDF toward fibroblasts and endothelial cells may serve to program an ordered temporal sequence of scaffold building followed by angiogenesis during wound healing. J. Cell. Biochem. 104: 1793–1802, 2008.

Up-regulating sphingosine 1-phosphate receptor-2 signaling impairs chemotactic, wound-healing, and morphogenetic responses in senescent endothelial cells.

Vascular endothelial cells (ECs) have a finite lifespan when cultured in vitro and eventually enter an irreversible growth arrest state called “cellular senescence.” It has been shown that sphingolipids may be involved in senescence; however, the molecular links involved are poorly understood. In this study, we investigated the signaling and functions of sphingosine 1-phosphate (S1P), a serum-borne bioactive sphingolipid, in ECs of different in vitro ages. We observed that S1P-regulated responses are significantly inhibited and the S1P1-3 receptor subtypes are markedly increased in senescent ECs. Increased expression of S1P1 and S1P2 was also observed in the lesion regions of atherosclerotic endothelium, where senescent ECs have been identified in vivo. S1P-induced Akt and ERK1/2 activation were comparable between ECs of different in vitro ages; however, PTEN (phosphatase and tensin homolog deleted on chromosome 10) activity was significantly elevated and Rac activation was inhibited in senescent ECs. Rac activation and senescent-associated impairments were restored in senescent ECs by the expression of dominant-negative PTEN and by knocking down S1P2 receptors. Furthermore, the senescent-associated impairments were induced in young ECs by the expression of S1P2 to a level similar to that of in vitro senescence. These results indicate that the impairment of function in senescent ECs in culture is mediated by an increase in S1P signaling through S1P2-mediated activation of the lipid phosphatase PTEN.

Reevaluation of the phospholipid composition in membranes of adult human lenses by 31 P NMR and MALDI MS

The phospholipid composition of adult human lens membranes differs dramatically from that of any other mammalian membrane. Due to minimal cell turnover, cells in the nucleus of the human lens may be considered as the longest lived cells in our body. This work reassesses previous assignments of phospholipid (31)P NMR resonances in adult human lenses. The new assignments are based not only on chemical shifts but also on temperature coefficients. By addition of known phospholipids and examination by matrix-assisted laser desorption/ionization mass spectrometry, several misassigned resonances have been corrected. The revised composition reveals the possible presence of ceramide-1-phosphate and dihydroceramide-1-phosphate. Among glycerophospholipids, the most abundant one does not correspond to phosphatidylglycerol but may be due to the lysoform of alkyl-acyl analogs of phosphatidylethanolamine. Besides sphingophospholipids, adult human lens membranes contain significant amounts of ether (1-O-alkyl) glycerophospholipids and their corresponding lysoforms.

Influence of temperature on 31P NMR chemical shifts of phospholipids and their metabolites I. In chloroform–methanol–water

Spectral overlap of (31)P NMR resonances and the lack of reproducibility in chemical shifts corresponding to phospholipids in organic solvents challenge the accuracy of band assignments and quantification. To alleviate these problems, the use of temperature coefficients is proposed. Changes in temperature enable the resolution of overlapped resonances and provide a facile approach for the computation of temperature coefficients. The coefficients were evaluated for various glycero- and sphingo-phospholipids. Their values suggest that differences in H-bonding between the phosphate and the head groups are responsible for the changes of chemical shift with temperature. Among parent phospholipids, and in addition to sphingomyelin, the smallest temperature coefficient values (closest to zero) were observed for phosphatidylcholine, phosphatidylglycerol, dihydrosphingomyelin, and cardiolipin. The highest values were exhibited by phospholipids with protonated head groups, such as phosphatidylserine and phosphatidylethanolamine. The lowest and, in fact, negative values were measured for phospholipids with an exposed phosphate group: phosphatidic acid, ceramide-1-phosphate, and dihydroceramide-1-phosphate. Diacyl, alkyl-acyl, and alkenyl-acyl phospholipids with the same head group exhibited comparable coefficients but differed slightly in chemical shifts. Compared to their parent glycerophospholipids, all lyso analogs had greater temperature coefficients, possibly due to the presence of an extra OH capable of forming a H-bond with the phosphate group.

Ligand-induced nuclear translocation of S1P1 receptors mediates Cyr61 and CTGF transcription in endothelial cells

Sphingosine-1-phosphate (S1P) receptor subtype 1 (S1P1), a G-protein coupled receptor (GPCR), regulates many biological activities of endothelial cells (ECs). In this report, we show that S1P1 receptors are present in the nuclei of ECs by using various biochemical and microscopic techniques such as cellular fractionation, immunogold labeling, and confocal microscopic analysis. Live cell imaging showed that plasma membrane S1P1 receptors are rapidly internalized and subsequently translocated to nuclear compartment upon S1P stimulation. Utilizing membrane biotinylation technique further supports the notion that nuclear S1P1 receptors were internalized from plasma membrane S1P1 after ligand treatment. Moreover, nuclear S1P1 is able to regulate the transcription of Cyr61 and CTGF, two growth factors functionally important in the regulation of vasculature. Collectively, these data suggest a novel S1P–S1P1 signaling axis present in the nuclear compartment of endothelial cells, which may regulate biological responses of endothelium.

Transcriptional Profile of Immediate Response to Ionizing Radiation Exposure

Astronauts participating in long duration space missions are likely to be exposed to ionizing radiation associated with highly energetic and charged heavy particles. Previously proposed gene biomarkers for radiation exposure include phosphorylated H2A Histone Family, Member X (γH2AX), Tumor Protein 53 (TP53), and Cyclin-Dependent Kinase Inhibitor 1A (CDKN1A). However, transcripts of these genes may not be the most suitable biomarkers for radiation exposure due to a lack of sensitivity or specificity. As part of a larger effort to develop lab-on-a-chip methods for detecting radiation exposure events using blood samples, we designed a dose–course microarray study in order to determine coding and non-coding RNA transcripts undergoing differential expression immediately following radiation exposure. The main goal was to elicit a small set of sensitive and specific radiation exposure biomarkers at low, medium, and high levels of ionizing radiation exposure. Four separate levels of radiation were considered: 0 Gray (Gy) control; 0.3 Gy; 1.5 Gy; and 3.0 Gy with four replicates at each radiation level. This report includes raw gene expression data files from the resulting microarray experiments from all three radiation levels ranging from a lower, typical exposure than an astronaut might see (0.3 Gy) to high, potentially lethal, levels of radiation (3.0 Gy). The data described here is available in NCBIs Gene Expression Omnibus (GEO), accession GSE64375.