Search

Mesenchymal stromal cells (MSCs) are multipotent stem cells with immunosuppressive and trophic support functions. While MSCs from different sources frequently display a similar appearance in culture, they often show differences in their surface marker and gene expression profiles. Although bone marrow is considered the "gold standard" tissue to isolate classical MSCs (BM-MSC), MSC-like cells are currently also derived from more easily accessible extra-embryonic tissues such as the umbilical cord. In this study, we defined the best way to isolate MSCs from the Wharton's jelly of the human umbilical cord (WJ-MSC) and assessed the mesenchymal and immunological phenotype of BM-MSC and WJ-MSC. Moreover, the gene expression profile of established WJ-MSC cultures was compared to two different bone marrow-derived stem cell populations (BM-MSC and multipotent adult progenitor cells or MAPC ® ). We observed that explant culturing of Wharton's jelly matrix is superior to collagenase tissue digestion for obtaining mesenchymal-like cells, with explant isolated cells displaying increased expansion potential. While being phenotypically similar to adult MSCs, WJ-MSC show a different gene expression profile. Gene ontology analysis revealed that genes associated with cell adhesion, proliferation, and immune system functioning are enriched in WJ-MSC. In vivo transplantation confirms their immune modulatory effect on T cells, similar to BM-MSC and MAPC. Furthermore, WJ-MSC intrinsically overexpress genes involved in neurotrophic support and their secretome induces neuronal maturation of SH-SY5Y neuroblastoma cells to a greater extent than BM-MSC. This signature makes WJ-MSC an attractive candidate for cell-based therapy in neurodegenerative and immune-mediated central nervous system disorders such as multiple sclerosis, Parkinson's disease, or amyotrophic lateral sclerosis.

The recently discovered APOA5 gene has been shown in humans and mice to be important in determining plasma triglyceride levels, a major cardiovascular disease risk factor. apoAV represents the first described apolipoprotein where overexpression lowers triglyceride levels. Since fibrates represent a commonly used therapy for lowering plasma triglycerides in humans, we investigated their ability to modulate APOA5 gene expression and consequently influence plasma triglyceride levels. Human primary hepatocytes treated with Wy 14,643 or fenofibrate displayed a strong induction of APOA5 mRNA. Deletion and mutagenesis analyses of the proximal APOA5 promoter firmly demonstrate the presence of a functional peroxisome proliferator-activated receptor response element. These findings demonstrate that APOA5 is a highly responsive peroxisome proliferator-activated receptor alpha target gene and support its role as a major mediator for how fibrates reduce plasma triglycerides in humans.

Plasma fibrinogen levels have been identified as an important risk factor for cardiovascular diseases. Among the few compounds known to lower circulating fibrinogen levels in humans are certain fibrates. We have studied the regulation of fibrinogen gene expression by fibrates in rodents. Treatment of adult male rats with fenofibrate (0.5% [wt/wt] in the diet) for 7 days decreased hepatic Aalpha-, Bbeta-, and gamma-chain mRNA levels to 52% +/- 7%, 46% +/- 8%, and 81% +/- 19% of control values, respectively. In parallel, plasma fibrinogen concentrations were decreased to 63% +/- 7% of controls. The suppression of fibrinogen expression was dose-dependent and was already evident after 1 day at the highest dose of fenofibrate tested (0.5% [wt/wt]). Nuclear run-on experiments showed that the decrease in fibrinogen expression after fenofibrate occurred at the transcriptional level, as exemplified for the gene for the Aalpha-chain. Other fibrates tested showed similar effects on fibrinogen expression and transcription. The effect of fibrates is specific for peroxisome proliferator-activated receptor-alpha (PPARalpha) because a high-affinity ligand for PPARgamma, the thiazolidinedione BRL 49653, lowered triglyceride levels, but was unable to suppress fibrinogen expression. Direct evidence for the involvement of PPARalpha in the suppression of fibrinogen by fibrates was obtained using PPARalpha-null (-/-) mice. Compared with (+/+) mice, plasma fibrinogen levels in (-/-) mice were significantly higher (3.20 +/- 0.48 v 2.67 +/- 0.42 g/L). Also, hepatic fibrinogen Aalpha-chain mRNA levels were 25% +/- 11% higher in the (-/-) mice. On treatment with 0.2% (wt/wt) fenofibrate, a significant decrease in plasma fibrinogen to 77% +/- 10% of control levels and in hepatic fibrinogen Aalpha-chain mRNA levels to 65% +/- 12% of control levels was seen in (+/+) mice, but not in (-/-) mice. These studies show that PPARalpha regulates basal levels of plasma fibrinogen and establish that fibrate-suppressed expression of fibrinogen in rodents is mediated through PPARalpha.

Fibrates are widely used hypolipidemic drugs which activate the nuclear peroxisome proliferator-activated receptor (PPAR) alpha and thereby alter the transcription of genes controlling lipoprotein metabolism. Fibrates influence plasma high density lipoprotein and its major protein, apolipoprotein (apo) A-I, in an opposite manner in man (increase) versus rodents (decrease). In the present study we studied the molecular mechanisms of this species-specific regulation of apoA-I expression by fibrates. In primary rat and human hepatocytes fenofibric acid, respectively, decreased and increased apoA-I mRNA levels. The absence of induction of rat apoA-I gene expression by fibrates is due to 3 nucleotide differences between the rat and the human apoA-I promoter A site, rendering a positive PPAR-response element in the human apoA-I promoter nonfunctional in rats. In contrast, rat, but not human, apoA-I transcription is repressed by the nuclear receptor Rev-erbalpha, which binds to a negative response element adjacent to the TATA box of the rat apoA-I promoter. In rats fibrates increase liver Rev-erbalpha mRNA levels >10-fold. In conclusion, the opposite regulation of rat and human apoA-I gene expression by fibrates is linked to differences in cis-elements in their respective promoters leading to repression by Rev-erbalpha of rat apoA-I and activation by PPARalpha of human apoA-I. Finally, Rev-erbalpha is identified as a novel fibrate target gene, suggesting a role for this nuclear receptor in lipid and lipoprotein metabolism.

Hypertriglyceridemia is a metabolic complication of retinoid therapy. In this study, we analyzed whether retinoids increase the expression of apo C-III, an antagonist of plasma triglyceride catabolism. In men, isotretinoin treatment (80 mg/d; 5 d) resulted in elevated plasma apo C-III, but not apo E concentrations. In human hepatoma HepG2 cells, retinoids increased apo C-III mRNA and protein production. Transient transfection experiments indicated that retinoids increase apo C-III expression at the transcriptional level. This increased apo C-III transcription is mediated by the retinoid X receptor (RXR), since LG1069 (4-[1-(5,6,7,8-tetrahydro-3,5,5,8, 8-pentamethyl-2-naphtalenyl)ethenyl]benzoic acid), a RXR-specific agonist, but not TTNPB ((E)- 4-[2-(5,6,7,8-tetrahydro-5,5,8, 8-tetramethyl-2-naphtalenyl)propenyl]benzoic acid), a retinoic acid receptor (RAR)-specific agonist, induced apo C-III mRNA in HepG2 cells and primary human hepatocytes. Mutagenesis experiments localized the retinoid responsiveness to a cis-element consisting of two imperfect AGGTCA sequences spaced by one oligonucleotide (DR-1), within the previously identified C3P footprint site. Cotransfection assays showed that RXR, but not RAR, activates apo C-III transcription through this element either as a homo- or as a heterodimer with the peroxisome proliferator-activated receptor. Thus, apo C-III is a target gene for retinoids acting via RXR. Increased apo C-III expression may contribute to the hypertriglyceridemia and atherogenic lipoprotein profile observed after retinoid therapy.

Since elevated concentrations of plasma high density lipoprotein (HDL) and its major apolipoprotein (apo), apoA-I, confer protection against atherosclerosis, considerable research efforts have focussed on the identification of factors regulating apoA-I gene expression in an attempt to increase its production. Nuclear receptors are interesting candidates because they are transcription factors whose activity is ligand-dependent. In the present study we identified the orphan receptor RORalpha1 as an activator of apoA-I gene transcription. In apoA-I-expressing intestinal Caco-2 cells, overexpression of the RORalpha1, but not the RORalpha2 or RORalpha3 isoforms, increased rat apoA-I gene transcription. Deletion and site-directed mutagenesis experiments identified a functional ROR-responsive element (RORE) in the rat and mouse apoA-I gene promoters, which overlaps with the TATA box. Gel shift experiments indicated that this RORE binds the RORalpha1 isoform, but not the RORalpha2 or RORalpha3 isoforms. Furthermore, compared with wild type mice, apoA-I mRNA levels were significantly lower in small intestines of staggerer mice homozygous for a deletion in the RORalpha gene. In addition, reverse transcriptase-polymerase chain reaction analysis revealed the expression of RORalpha in small intestinal epithelium and in Caco-2 cells. These data indicate a novel, physiological role for RORalpha1 in the regulation of genes involved in lipid and lipoprotein metabolism and possibly in the development of metabolic diseases, such as atherosclerosis.