Gravity spun polycaprolactone fibers for applications in vascular tissue engineering:proliferation and function of human vascular endothelial cells

Poly(ε-caprolactone) (PCL) fibers produced by wet spinning from solutions in acetone under low-shear (gravity-flow) conditions resulted in fiber strength of 8 MPa and stiffness of 0.08 Gpa. Cold drawing to an extension of 500% resulted in an increase in fiber strength to 43 MPa and stiffness to 0.3 GPa. The growth rate of human umbilical vein endothelial cells (HUVECs) (seeded at a density of 5 × 104 cells/mL) on as-spun fibers was consistently lower than that measured on tissue culture plastic (TCP) beyond day 2. Cell proliferation was similar on gelatin-coated fibers and TCP over 7 days and higher by a factor of 1.9 on 500% cold-drawn PCL fibers relative to TCP up to 4 days. Cell growth on PCL fibers exceeded that on Dacron monofilament by at least a factor of 3.7 at 9 days. Scanning electron microscopy revealed formation of a cell layer on samples of cold-drawn and gelatin-coated fibers after 24 hours in culture. Similar levels of ICAM-1 expression by HUVECs attached to PCL fibers and TCP were measured using RT-PCR and flow cytometry, indicative of low levels of immune activation. Retention of a specific function of HUVECs attached to PCL fibers was demonstrated by measuring their immune response to lipopolysaccharide. Levels of ICAM-1 expression increased by approximately 11% in cells attached to PCL fibers and TCP. The high fiber compliance, favorable endothelial cell proliferation rates, and retention of an important immune response of attached HUVECS support the use of gravity spun PCL fibers for three-dimensional scaffold production in vascular tissue engineering. © Mary Ann Liebert, Inc.

Mouse splenocyte proliferation and its modulation by sex steroids

Concanavalin A, a T cell mitogen enhanced DNA synthesis in murine splenocytes. Amongst the early signals prior to this event was an increase in cytosolic calcium derived from both intra- and extracellular sources. The requirements for extracellular calcium persisted for four hours after the lectin administration which itself was needed for six hours. Putative calcium channel antagonists and calmodulin inhibitors blocked ihe increase in DNA synthesis. The calcium signal was mimicked by application of the ionophore, A23187, although no increase in DNA synthesis occurred. An activator of protein kinase C, 12-0- tetradecanoylphorbol 13-acetate, had little effect in isolation but the combined application of these two agents greatly enhanced DNA synthesis. The natural mediators of these events are presumed to be inositol trisphosphate and diacylglycerol derived from phosphatidylinositol bisphosphate hydrolysis. Lectin application and protein kinase C activation both increased intracellular pH possibly as a result of Na'l'/H"'' exchange since amiloride an inhibitor of this antiporter inhibited lectin induced DNA synthesis. The calcium and hydrogen ionic changes occur within minutes of lectin application; the protracted requirement for this mitogen suggests further signalling mechanisms occur to elicit maximum DNA synthesis in these cells. Gonadectomy caused an increase in thymic and splenic weight. Spleno-cytes derived from castrated mice showed no change in mitogen response whereas those from ovariectomised mice demonstrated a reduced lectin sensitivity. Testosterone, 5 a dihydrotestosterone, a and 0 oestradiol all inhibited lectin induced DNA synthesis but only at pharmacological concentrations. Testosterone glucuronide and cholesterol were without effect Studies with mouse serum fractions of differing steroidal status were unable to confirm the presence or absence of serum factors which might mediate the effects of steroid on lymphoid cells, all fractions tested inhibited lymphocyte transformation. Both interleukin-2 and lipopolysaccharide induced splenocyte mitogene-sis was also impaired by high steroid concentrations in vitro, suggesting that steroids mediate their effect by a non-specific, non-receptor-mediated event.

The role of insulin and the insulin-like growth factors in the proliferation of the rat thymic lymphocyte

Concanavalin A, provoked a 35-fold increase in the rate of proliferation of rat thymocytes. Insulin (10-6M), and insulin-like growth factor I (10-10M) approximately doubled the rate of DNA synthesis. Both of these structurally related molecules acted through the type I insulin-like growth factor receptor. The sequential addition of Concanavalin A and insulin, promoted a much greater proliferative response than to either of the two agonists alone. Insulin also increased the uptake of glucose and amino acids by the cells. Glucose uptake was enhanced at insulin concentrations of 10-6M and 10-10M. Amino acid uptake was more strongly affected at the higher concentration. Insulin-like growth factor I (10-11M) also enhanced amino acid uptake. The effects of insulin on metabolism were mediated by both insulin and type I insulin-like growth factor receptors. These effects were greatly enhanced after a pre-treatment with Concanavalin A. Concanavalin A provided a primary mitogenic signal to the cells. Amongst the responses was an increased expression of insulin and/or type I insulin-like growth factor receptors. The consequent enhanced cellular sensitivity to these agonists, enabled them to facilitate the passage of the cells through the cell cycle by: i) providing a secondary mitogenic signal, and ii) promoting the uptake of raw materials and energy substrates. The initiation of DNA synthesis and passage through the cell cycle was thus punctuated by the sequential expression of various cell surface receptors. This regulated cellular sensitivity, enabling them to react in a precisely orchestrated fashion to hormones and other molecules in their environment. The intracellular mechanism of insulin action remains an enigma. Although the presence of extracellular calcium was essential for insulin stimulation of amino acid uptake and DNA synthesis, the cation did not subserve a direct mediator function. Insulin promoted an increase in intracellular pH, which was mediated by the Na+/H+ antiport. Other mechanisms were probably also involved in mediating the full cellular response to insulin.

Methylglyoxal, glyoxalses and cell proliferation

The metabolic function of the glyoxalase system was investigated in (a) the differentiation and proliferation of human tumour cells in vitro, (b) the cell-free assembly of microtubules and (c) in the red blood cells during hyperglycaemia associated with Diabetes Mellitus. Chemically-induced differentiation of human promyelocytic HL60 leukaemia cells to neutrophils, and K562 erythroleukaemia cells, was accompanied by a decrease and an increase in the activity of glyoxalase I, respectively. Growth-arrest of Burkitt's lymphoma Raji cells and GM892 lymphoblastoid cells was accompanied by an increase and a decrease in the activity of glyoxalase I respectively. However, differentiation and growth arrest generally proceeded with an increase in the activity of glyoxalase II. Glyoxalase I activity did not consistently correlate with cell differentiation or proliferation status; hence, it is unlikely that glyoxalase I activity is either an indicator or a regulator of cell differentiation or proliferation. Conversely, glyoxalase II activity consistently increased during cell differentiation and growth-arrest and may be both an indicator and regulator of cell differentiation or proliferation. This may be related to the control of cellular microtubule assembly. S-D-Lactoylglutathione potentiated the cell-free, GTP-promoted assembly of microtubules. The effect was dose-related and was inhibited by glyoxalase II. During assembly, S-D-lactoylglutathione was consumed. This suggests that the glyoxalase system, through the influence of S-D-lactoylglutathione, may regulate the assembly of microtubules in cellular systems The whole blood concentrations of methylglyoxal and S-D-lactoylglutathione were increased in Diabetes Mellitus. There was no significant difference between red blood cell glyoxalase activities in diabetics, compared to healthy controls. However, insulin-dependent diabetic patients with retinopathy had a significantly higher glyoxalase I activity and a lower glyoxalase II activity, than patients without retinopathy. Diabetic retinopathy correlated with high glyoxalase I activity and low glyoxalase II activity and suggests the glyoxalase system may be involved in the development of diabetic complications.

Control of proliferation in the rat thymus

Quiescent rat thymocytes were stimulated to divide by a variety of agents. One such mitogen was the neurotransmitter acetylcholine which exhibited a biphasic action. Interaction with low affinity nicotinic receptors was linked with an obligatory requirement for magnesium ions whereas combination with high affinity muscarinic receptors induced mitosis only if calcium ions were present in the medium. Binding of acetylcholine to its muscarinic receptor enhanced calcium influx and increased intracellular calcium levels causing calmodulin activation, a necessary prelude to DNA synthesis and mitosis. Nicotinic receptor activation may be associated with a magnesium influx and stimulation of cells in a calmodulin-independent fashion. Parathyroid hormone and its analogues exhibited only a monophasic mitogenic action. This response was linked to calcium influx, a rise in cytosolic calcium and calmodulin activation. Parathyroid hormone did not stimulate adenylate cyclase in thymocytes and decreased cellular cyclic AMP concentrations. Picomolar amounts of interleukin-2 (IL-2) also stimulated division in thymocytes derived from 3-month old rats by binding to high affinity receptors. The response in thymocytes from newborn and foetal animals was greater reflecting the larger proportion of cells bearing receptors at this age. The mitogenic effect of IL-2 was abolished by a monoclonal antibody directed against the IL-2 receptor. Injections of IL-2 itself or the administration of IL-2 secreting activated syngeneic spleen cells also stimulated proliferation of both thymus and bone marrow cells in vivo. Likewise immunisation with pertussis toxin, which enhances endogenous IL2 production, also increased mitosis in these tissues. Calcium influx, increased cytosolic Ca2+ levels and calmodulin activation are associated features of the mitogenic action of IL-2. Interleukin-1 was also found to be mitogenic in thymic lymphocyte cultures. The responses to this mitogen and to parathyroid hormone and acetylcholine were not inhibited by the anti-IL2 receptor antibody suggesting that the thymic lymphocyte bears discrete receptors for these agents. Subtle interactions of hormones, neurotransmitters and interleukins may thus contribute to the turnover and control of lymphoid cells in the thymus and perhaps bone-marrow.

Vascular endothelial growth factor-induced endothelial cell proliferation is regulated by interaction between VEGFR-2, SH-PTP1 and eNOS

VEGF receptor-2 plays a critical role in endothelial cell proliferation during angiogenesis. However, regulation of receptor activity remains incompletely explained. Here, we demonstrate that VEGF stimulates microvascular endothelial cell proliferation in a dose-dependent manner with VEGF-induced proliferation being greatest at 5 and 100 ng/ml and significantly reduced at intermediate concentrations (>50% at 20 ng/ml). Neutralization studies confirmed that signaling occurs via VEGFR-2. In a similar fashion, ERK/MAPK is strongly activated in response to VEGF stimulation as demonstrated by its phosphorylation, but with a decrease in phosphoryation at 20 ng/ml VEGF. Immunoblotting analysis revealed that VEGF did not cause a dose-dependent change in expression of VEGFR-2 but instead resulted in reduced phosphorylation of VEGFR-2 when cells were exposed to 10 and 20 ng/ml of VEGF. VEGFR-2 dephosphorylation was associated with an increase in the protein tyrosine phosphatase, SH-PTP1, and endothelial nitric oxide synthase (eNOS). Immunoprecipitation and selective immunoblotting confirmed the association between VEGFR-2 dephosphorylation and the upregulation of SH-PTP1 and eNOS. Transfection of endothelial cells with antisense oligonucleotide against VEGFR-2 completely abolished VEGF-induced proliferation, whereas anti SH-PTP1 dramatically increased VEGF-induced proliferation by 1 and 5-fold at 10 and 200 ng/ml VEGF, respectively. Suppression of eNOS expression only abolished endothelial cell proliferation at VEGF concentrations above 20 ng/ml. Taken together, these results indicate that activation of VEGFR-2 by VEGF enhances SH-PTP1 activity and eNOS expression, which in turn lead to two diverse events: one is that SH-PTP1 dephosphorylates VEGFR-2 and ERK/MAPK, which weaken VEGF mitogenic activity, and the other is that eNOS increases nitric oxide production which in turn lowers SH-PTP1 activity via S-nitrosylation.

Phytoestrogen-ind uced glucose uptake and cellular proliferation in L6 myotubesadipocyte function

Aims: Oestrogens are known to act on a number of tissues throughout the body via classical oestrogen receptors, alpha (ER-a) and beta (ER-beta). Previous research has shown that oestrogens can regulate skeletal muscle glucose uptake cellular proliferation. Thus, oestrogens and related molecules provide an interesting focus for research into possible therapies for the treatment of metabolic disorders and sarcopenia. Enterodiol and enterolactone are plant derived mammalian enterolignans which share a struc- tural similarity to the human oestrogen oestradiol. Methods: In the present study we incubated the differentiated rat skeletal muscle cell line L6 concentration ranges of both com- pounds in the presence/absence of oestrogen receptor antagonists and measured glucose uptake using the non-metabolised glucose analogue 2-NBDG. Cellular proliferation was also measured using a modified MTS assay. Results: Enterolactone was seen to cause a significant increase in cellular proliferation after 48h (a maximal 25% at 0.1nmol/l), in an ER-a dependent mechanism. Incubation with 10nmol/l and 100nmol/l enterodiol caused significant increases in 2-NBDG (5000% compared with control, p < 0.001) and 2h glucose depletion from media (15% increase compared with control, p < 0.05), also in an ER-a dependent way. These results suggest these dietary derived oestrogen-like molecules might be of potential use in targeting metabolic disorders or sarcopenia. Conclusion: We can report here that the phytoestrogen derived molecules enterodiol and enterolactone interact with ER-a in the myotubes to regulate glucose uptake and cellular proliferation respectively.