Undifferentiated human mesenchymal stem cells (hMSCs) are highly sensitive to mechanical strain: transcriptionally controlled early osteo-chondrogenic response in vitro

SummaryObjectivePhysical cues play a crucial role in skeletogenesis and osteochondral regeneration. Although human mesenchymal stem cells (hMSCs) offer considerable therapeutic potential, little is known about the molecular mechanisms that control their differentiation. We hypothesized that mechanical strain might be an inherent stimulus for chondrogenic and/or osteogenic differentiation in undifferentiated hMSCs, where c-Fos (FOS) might play a major role in mechanotransduction.MethodhMSCs from 10 donors were intermittently stimulated by cyclic tensile strain (CTS) at 3000 μstrain for a period of 3 days. Differential gene expression of strained and unstrained hMSCs was analysed by real-time RT-PCR for several marker genes, including the transcription factors FOS, RUNX2, SOX9, and others. Additionally, alkaline phosphatase activity (ALP) was determined kinetically.ResultsThe application of CTS significantly stimulated the expression levels of the early chondrogenic and osteogenic marker genes (SOX9, LUM, DCN; RUNX2, SPARC, SPP1, ALPL); this was accompanied by stimulation of ALP activity (+38%±12 standard error of mean, P<0.05). Matrix analysis revealed that the osteo-chondrogenic response followed a coordinated expression pattern, in which FOS was attributed to early osteogenic but not chondrogenic differentiation.ConclusionUndifferentiated hMSCs are highly sensitive to mechanical strain with a transcriptionally controlled osteo-chondrogenic differentiation response in vitro

High-Level Expression of Various Apolipoprotein (a) Isoforms by "Transferrinfection". The Role of Kringle IV Sequences in the Extracellular Association with Low-Density Lipoprotein

Characterization of the assembly of lipoprotein(a) [Lp(a)] is of fundamental importance to understanding the biosynthesis and metabolism of this atherogenic lipoprotein. Since no established cell lines exist that express Lp(a) or apolipoprotein(a) [apo(a)], a "transferrinfection" system for apo(a) was developed utilizing adenovirus receptor- and transferrin receptor-mediated DNA uptake into cells. Using this method, different apo(a) cDNA constructions of variable length, due to the presence of 3, 5, 7, 9, 15, or 18 internal kringle IV sequences, were expressed in cos-7 cells or CHO cells. All constructions contained kringle IV-36, which includes the only unpaired cysteine residue (Cys-4057) in apo(a). r-Apo(a) was synthesized as a precursor and secreted as mature apolipoprotein into the medium. When medium containing r-apo(a) with 9, 15, or 18 kringle IV repeats was mixed with normal human plasma LDL, stable complexes formed that had a bouyant density typical of Lp(a). Association was substantially decreased if Cys-4057 on r-apo(a) was replaced by Arg by site-directed mutagenesis or if Cys-4057 was chemically modified. Lack of association was also observed with r-apo(a) containing only 3, 5, or 7 kringle IV repeats without "unique kringle IV sequences", although Cys-4057 was present in all of these constructions. Synthesis and secretion of r-apo(a) was not dependent on its sialic acid content. r-Apo(a) was expressed even more efficiently in sialylation-defective CHO cells than in wild-type CHO cells. In transfected CHO cells defective in the addition of N-acetylglucosamine, apo(a) secretion was found to be decreased by 50%. Extracellular association with LDL was not affected by the carbohydrate moiety of r-apo(a), indicating a protein-protein interaction between r-apo(a) and apoB. These results show that, besides kringle IV-36, other kringle IV sequences are necessary for the extracellular association of r-apo(a) with LDL. Changes in the carbohydrate moiety of apo(a), however, do not affect complex formation

Familial Study on "Sinking pre-beta", the Lp(a) Lipoprotein, and its Relationship with Serum Lipids, Apolipoprotein A-I and B and Clinical Atherosclerosis

Peer Reviewe

Selective delipidation of plasma HDL enhances reverse cholesterol transport in vivo

Uptake of cholesterol from peripheral cells by nascent small HDL circulating in plasma is necessary to prevent atherosclerosis. This process, termed reverse cholesterol transport, produces larger cholesterol-rich HDL that transfers its cholesterol to the liver facilitating excretion. Most HDL in plasma is cholesterol-rich. We demonstrate that treating plasma with a novel selective delipidation procedure converts large to small HDL [HDL-selectively delipidated (HDL-sdl)]. HDL-sdl contains several cholesterol-depleted species resembling small alpha, prebeta-1, and other prebeta forms. Selective delipidation markedly increases efficacy of plasma to stimulate ABCA1-mediated cholesterol transfer from monocytic cells to HDL. Plasma from African Green monkeys underwent selective HDL delipidation. The delipidated plasma was reinfused into five monkeys. Prebeta-1-like HDL had a plasma residence time of 8 +/- 6 h and was converted entirely to large alpha-HDL having residence times of 13-14 h. Small alpha-HDL was converted entirely to large alpha-HDL. These findings suggest that selective HDL delipidation activates reverse cholesterol transport, in vivo and in vitro. Treatment with delipidated plasma tended to reduce diet-induced aortic atherosclerosis in monkeys measured by intravascular ultrasound. These findings link the conversion of small to large HDL, in vivo, to improvement in atherosclerosis