Effective seeding of smooth muscle cells into tubular poly(trimethylene carbonate) scaffolds for vascular tissue engineering.

Porous tubular poly(trimethylene carbonate) (PTMC) scaffolds for vascular tissue engineering, with an inner diameter of 3 mm and a wall thickness of 1 mm, were prepared by means of dip-coating and subsequent leaching of NaCl particles. The scaffolds, with an average pore size of 110 µm and a porosity of 85%, showed a smooth muscle cell (SMC) seeding efficiency of only 10%. To increase the efficiency of cell seeding, these scaffolds were coated with a microporous PTMC outer layer with a thickness of 0.1-0.4 mm, an average pore size of 28 µm, and a porosity of 65%. Coating of the scaffolds with the microporous outer layer did not influence the inner pore structure or the mechanical properties of the scaffolds to a significant extent. The intrinsic permeability of the scaffolds decreased from 60 × 10(-10) m(2) to approximately 5 × 10(-10) m(2) after coating with the microporous outer layer. The latter value is still relatively high indicating that these scaffolds may facilitate sufficient diffusion of nutrients and waste products during cell culturing. The efficiency of SMC seeding determined after 24 h cell adhesion in the scaffolds increased from less than 10% to 43% after coating with the microporous outer layer. The cells were homogeneously distributed in the scaffolds and cell numbers increased 60% during culturing for 7 days under stationary conditions. It is concluded that coating of porous tubular PTMC scaffolds with a microporous PTMC outer layer facilitates effective cell seeding in these scaffolds.

Flexible and elastic porous poly(trimethylene carbonate) structures for use in vascular tissue engineering.

Biocompatible and elastic porous tubular structures based on poly(1,3-trimethylene carbonate), PTMC, were developed as scaffolds for tissue engineering of small-diameter blood vessels. High-molecular-weight PTMC (M(n) = 4.37 x 10(5)) was cross-linked by gamma-irradiation in an inert nitrogen atmosphere. The resulting networks (50-70% gel content) were elastic and creep resistant. The PTMC materials were highly biocompatible as determined by cell adhesion and proliferation studies using various relevant cell types (human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) and mesenchymal stem cells (MSCs)). Dimensionally stable tubular scaffolds with an interconnected pore network were prepared by particulate leaching. Different cross-linked porous PTMC specimens with average pore sizes ranging between 55 and 116 microm, and porosities ranging from 59% to 83% were prepared. These scaffolds were highly compliant and flexible, with high elongations at break. Furthermore, their resistance to creep was excellent and under cyclic loading conditions (20 deformation cycles to 30% elongation) no permanent deformation occurred. Seeding of SMCs into the wall of the tubular structures was done by carefully perfusing cell suspensions with syringes from the lumen through the wall. The cells were then cultured for 7 days. Upon proliferation of the SMCs, the formed blood vessel constructs had excellent mechanical properties. Their radial tensile strengths had increased from 0.23 to 0.78 MPa, which is close to those of natural blood vessels.

Lowering caveolin-1 expression in human vascular endothelial cells inhibits signal transduction in response to shear stress.

Vascular endothelial cells have an extensive response to physiological levels of shear stress. There is evidence that the protein caveolin-1 is involved in the early phase of this response. In this study, caveolin-1 was downregulated in human endothelial cells by RNAi. When these cells were subjected to a shear stress of 15 dyn/cm(2) for 10 minutes, activation of Akt and ERK1/2 was significantly lower than in control cells. Moreover, activation of Akt and ERK1/2 in response to vascular endothelial growth factor was significantly lower in cells with low levels of caveolin-1. However, activation of integrin-mediated signaling during cell adhesion onto fibronectin was not hampered by lowered caveolin-1 levels. In conclusion, caveolin-1 is an essential component in the response of endothelial cells to shear stress. Furthermore, the results suggest that the role of caveolin-1 in this process lies in facilitating efficient VEGFR2-mediated signaling.

Effect of conjugated linoleic acid supplementation after weight loss on appetite and food intake in overweight subjects.

OBJECTIVE: To study the effects of 13 weeks conjugated linoleic acid (CLA) supplementation in overweight subjects on body-weight maintenance, parameters of appetite and energy intake (EI) at breakfast after weight loss. DESIGN: This study had a double-blind, placebo-controlled randomized design. SUBJECTS: A total of 26 men and 28 women (age 37.8+/-7.7 y; body mass index 27.8+/-1.5 kg/m(2)). INTERVENTIONS: Subjects were first submitted to a very-low-calorie diet (VLCD; 2.1 MJ/day) for 3 weeks after which they started with the 13-week intervention period. They either received 1.8 g CLA or placebo per day or 3.6 g CLA or placebo per day. Additionally, subjects of the high dosage intervention replaced their habitual lunch by one meal of a protein-rich, low-energy supplement. EI was measured at breakfast and appetite profile after an overnight fast. RESULTS: The mean body weight loss was 6.9+/-1.7% of their original body weight. Multiple regression analysis showed that at the end of the 13-week intervention, CLA did not have an effect on body weight regain. Feelings of fullness and satiety were increased and feelings of hunger were decreased after 13 weeks intervention by CLA compared to placebo, independent of %body weight regain. However, EI measured at breakfast was not affected by CLA. CONCLUSION: Appetite (hunger, satiety and fullness) was favorably, dose-independently affected by a 13-week consumption of 1.8 or 3.6 g CLA/day. This did not result in a lower EI at breakfast or an improved body-weight maintenance after weight loss.

The effect of conjugated linoleic acid supplementation after weight loss on body weight regain, body composition, and resting metabolic rate in overweight subjects.

OBJECTIVE: To study the effects of 13 weeks conjugated linoleic acid (CLA) supplementation in overweight subjects after weight loss on weight regain, body composition, resting metabolic rate, substrate oxidation, and blood plasma parameters. DESIGN: This study had a double-blind, placebo-controlled randomized design. Subjects were first submitted to a very-low-calorie diet (VLCD 2.1 MJ/d) for 3 weeks after which they started with the 13-week intervention period. They either received 1.8 g CLA or placebo per day (low dosage, LD) or 3.6 g CLA or placebo per day (high dosage, HD). SUBJECTS: A total of 26 men and 28 women (age 37.8+/-7.7 y; body mass index (BMI) 27.8+/-1.5 kg/m(2)). MEASUREMENTS: Before VLCD (t=-3), after VLCD but before CLA or placebo intervention (t=0) and after 13-week CLA or placebo intervention (t=13), body weight, body composition (hydrodensitometry and deuterium dilution), resting metabolic rate, substrate oxidation, physical activity, and blood plasma parameters (glucose, insulin, triacylglycerol, free fatty acids, glycerol and beta-hydroxy butyrate) were measured. RESULTS: The VLCD significantly lowered body weight (6.9+/-1.7%), %body fat, fat mass, fat-free mass, resting metabolic rate, respiratory quotient and plasma glucose, insulin, and triacylglycerol concentrations, while free fatty acids, glycerol and beta-hydroxy butyrate concentrations were increased. Multiple regression analysis showed that at the end of the 13-week intervention, CLA did not affect %body weight regain (CLA LD 47.9+/-88.2%, CLA HD 27.4+/-29.8%, Placebo LD 32.0+/-42.8%, Placebo HD 22.5+/-37.9%). The regain of fat-free mass was increased by CLA (LD 6.2+/-3.9, HD 4.6+/-2.4%) compared to placebo (LD 2.8+/-3.2%, HD 3.4+/-3.6%), independent of %body weight regain and physical activity. As a consequence of an increased regain of fat-free mass by CLA, resting metabolic rate was increased by CLA (LD 12.0+/-11.4%, HD 13.7+/-14.4%) compared to placebo (LD 9.1+/-11.0%, HD 8.6+/-8.5%). Substrate oxidation and blood plasma parameters were not affected by CLA. CONCLUSION: In conclusion, the regain of fat-free mass was favorably, dose-independently affected by a 13-week consumption of 1.8 or 3.6 g CLA/day and consequently increased the resting metabolic rate. However, it did not result in improved body weight maintenance after weight loss.

Chemosensitization of myeloma plasma cells by an antisense-mediated downregulation of Bcl-2 protein.

An antisense oligodeoxynucleotide (ODN) complementary to the first six codons of the Bcl-2 mRNA, G3139 (oblimersen sodium; Genasense), has been shown to downregulate Bcl-2 and produce responses in a variety of malignancies including drug-resistant lymphoma. Incubation of ex vivo purified plasma cells from patients with multiple myeloma (MM) with carboxyfluorescein (FAM)-labeled antisense ODNs resulted in a time- and dose-dependent uptake in the cytoplasm and nucleus. No major differences in uptake of Bcl-2 antisense ODNs were observed among patients' samples. Incubation of purified myeloma plasma cells with G3139, but not solvent or reverse polarity control ODNs, resulted in a reduction (>75%) of Bcl-2 mRNA levels after 2 and 4 days, as measured by Real-Time PCR. Treatment with G3139 led to a sequence-specific reduction of Bcl-2 protein levels within 4 days of exposure in 10 out of 11 clinical samples from patients with chemosensitive and multidrug-resistant disease, without significant reduction of alpha-Actin, Bax, Bcl-XL, or Mcl-1 proteins. This resulted in a significantly enhanced sensitivity of the myeloma tumor cells to dexamethasone or doxorubicin-induced apoptosis. G3139 can consistently enter myeloma cells, downregulate the expression of Bcl-2, and enhance the efficacy of myeloma therapy. These data support further clinical evaluation of G3139 therapy in multiple myeloma.

The cholesterol lowering drug lovastatin induces cell death in myeloma plasma cells.

Lovastatin is an irreversible inhibitor of HMG-CoA reductase and blocks the production of mevalonate, a critical compound in the production of cholesterol and isoprenoids. Isoprenylation of target proteins, like the GTP-binding protein Ras, is essential for their membrane localization and subsequent participation in intracellular signaling cascades. Lovastatin effectively decreased the viability of plasma cells from cell lines (n = 10) and myeloma patients' samples (n = 8) in a dose- and time-dependent way. Importantly, co-incubation of lovastatin with dexamethasone had a synergistic effect in inducing plasma cell cytotoxity. This effect was not the consequence of a change in the protein expression levels of Bcl-2 or Bax induced by lovastatin. The decrease in plasma cell viability was the result of induction of apoptosis and inhibition of proliferation. Mevalonate effectively reversed the cytotoxic and cytostatic effects of lovastatin in plasma cells. The cytotoxic activity of lovastatin was higher in Pgp expressing cell lines, but did not correlate with the multidrug resistance (MDR)-related proteins LRP, Bcl-2 and Bax. Lovastatin treatment resulted in a shift of Ras localization from the membrane to the cytosol that was reversed by mevalonate. The data presented in this paper warrant study of lovastatin alone or in combination with therapeutic drugs, in the treatment of myeloma patients.