Effect of maturation on the osteogenic response of cultured stromal bone marrow cells to basic fibroblast growth factor.

Formation of bone-like tissue in culture by stromal bone marrow cells (SBMC) derived from young growing rats is dependent on dexamethasone (Dex) (Cell Tissue Res 254:317; 1988) and is significantly enhanced by basic fibroblast growth factor (bFGF) (J Bone Miner Res 8:919; 1993). The aim of this study was to examine the effect of maturation on the osteogenic potential and the response to Dex and bFGF of SBMC by using cultures derived from young growing (6 weeks old) and adult (9 months old) rats. SBMC cultures were grown in the presence of Dex (10(-8) or 10(-7) mol/L) at both P(0) and P(1) and either in the presence or absence of bFGF. The effect of Dex and bFGF on mineralized bone-like tissue (MBT) formation was assessed at P(1). The highest levels of mineralized tissue formation in P(1) subcultures in the absence of bFGF were obtained when cultures derived from young rats (6 weeks old) were treated with Dex 10(-7) and 10(-8) mol/L at P(0) and P(1), respectively, and when cultures derived from adult rats were exposed to Dex 10(-8) mol/L both at P(0) and P(1). Under these optimal Dex concentrations, the amount of MBT formed by adult rat-derived cultures was 15-fold lower than that of young rat-derived ones. The addition of bFGF to P(0) cultures or to P(1) cultures grown under optimal Dex conditions enhanced MBT formation in P(1) cultures derived from both young and adult rats, but this effect was considerably more pronounced in the adult rat-derived cultures. The maximal levels of MBT formation were produced by cultures derived from adult rats treated with bFGF at both P(0) and P(1), whereas in cultures derived from young rats, the addition of bFGF at P(0) was not necessary for maximal MBT production. This stimulating effect of bFGF on MBT formation by adult rat-derived cultures was accompanied by a 2.2-, 1.8-, and 4.3-fold increase in proliferation, alkaline phosphatase activity, and Ca(2+) deposition rate, respectively. bFGF increased the level of glucocorticoid receptor by approximately 2. 3-fold in Dex-treated cultures derived from young animals. These results indicate that maturation is associated with a decrease in the proportion of osteoprogenitor cells in the stromal bone marrow and in their capacity to express the osteogenic phenotype. They further point to the significant role of bFGF in stimulating proliferation and osteogenic expression of stromal bone marrow osteoprogenitors derived from adult rats.

Shear-induced platelet adhesion and aggregation on subendothelium are increased in diabetic patients.

Increased platelet aggregation has been suggested to play a role in the accelerated atherosclerosis of diabetics. However the physiological relevance of the aggregation tests has been questioned. The purpose of this study was to determine platelet activation in diabetic patients, using a novel device--the cone and plate(let) analyzer--to measure shear-induced platelet adhesion and aggregation on extracellular matrix (ECM). Whole blood platelet adhesion and aggregation in patients with noninsulin-dependent diabetes mellitus (n=82) and in nondiabetic controls (n=71) were compared. Clinical and laboratory characteristics of the diabetic patients were analyzed for possible correlation with parameters of platelet activity. Patients with diabetes had a significantly increased platelet activation compared to nondiabetic subjects, demonstrated by an increased adhesion to the ECM (surface coverage, 23% [95% confidence interval, 22-25%] vs. 19% [95% confidence interval, 18-20%], respectively) and an increased average size of the ECM-bound aggregates (54 microm2 [95% confidence interval, 51-57 microm2] vs. 47 microm2 [95% confidence interval, 43-51 microm2], respectively). Platelet adhesion in the diabetic group was found to correlate with triglyceride levels (r=0.36) and hematocrit values (r=0.31) and inversely with high-density lipoprotein cholesterol levels (r=0.30). There were no correlation, however, between parameters of platelet reactivity and duration of diabetes, vascular complications and low-density lipoprotein levels. Our data demonstrate an increased platelet adhesion and aggregation in diabetic patients and suggest a modulatory role of diabetic dyslipidemia.

Oxidation decreases low density lipoprotein association with the subendothelium extracellular matrix.

Atherosclerosis is initiated by accumulation of low density lipoprotein (LDL) in the subendothelium extracellular matrix (ECM) followed by oxidation and scavenger receptor mediated uptake by the vessel wall recruited macrophages. The effect of oxidation on LDL association with the ECM is not yet clear. In the present study we examined the hypothesis that excessive oxidation of LDL results in decreased LDL association with ECM, thereby increasing its accessibility to the scavenger receptor on macrophages. We have studied the relative association of Cu+2 ion oxidized LDL to native LDL with the native or oxidized bovine corneal endothelial cells ECM. Oxidation of LDL decreased its binding to the ECM. The kinetic of this process was characterized by approximately 1 h lag phase followed by a significant decreased binding of 80% after 6.5 h oxidation. This kinetic more closely resembled the pattern of the oxidation product dienals accumulation rather than thiobarbituric acid reactive substance formation. Oxidation of ECM-bound LDL resulted in an increased LDL release from the ECM as a function of Cu+2 ion concentration with a maximal increase of 2-fold at 3 microM. ECM oxidation prior to exposure to LDL resulted in a 30% decrease in LDL binding to the ECM. In conclusion, these results suggest that oxidation processes in the vessel wall result in increased dissociation of ECM-bound LDL, which in turn makes this oxidized LDL more accessible for binding and uptake by macrophages leading to foam cell formation.

A new method for quantitative analysis of whole blood platelet interaction with extracellular matrix under flow conditions.

A new method and device in which whole blood platelet deposition and aggregation on extracellular matrix (ECM) under defined shear conditions is quantitatively evaluated was developed. A 0.25 mL aliquot of citrated whole blood is placed on ECM and a defined shear rate is applied for 2 min using a cone and plate device. This is followed by staining and measuring the number of stained objects, the percentage of ECM surface covered with stained objects and the average size of the objects using an image analyzer. When normal blood is analyzed, platelet deposition is a shear and a time dependent process, reaching maximal levels within 2 min at high shear rate (1300 s-1) of about 20% surface coverage and average aggregate size of about 40-50 microns 2. These two parameters demonstrated positive correlation with the platelet count and the hematocrit. Studies using samples from patients with von Willebrand disease (vWD) and Glanzmann's Thrombasthenia (GT) were performed and demonstrated the ability of the new method to detect these pathological conditions. Blood samples of vWD patients showed a very low adhesion and aggregation at high shear rate as reflected by very low surface coverage (5.2%) and average particle size of single platelets (21.3 microns2). GT samples at a high shear rate demonstrated surface coverage similar to normal blood samples (21.7%) but with average particle size of single platelets (21.3 microns2). The new method is an alternative method to clinically evaluate platelet function under close to physiological conditions.

Effect of basic fibroblast growth factor on the growth and differentiation of adult stromal bone marrow cells: enhanced development of mineralized bone-like tissue in culture.

Rat stromal bone marrow cells (SBMC) were shown to produce mineralized bone-like tissue in culture in the presence of dexamethasone, ascorbic acid, and beta-glycerophosphate. The addition of 3 ng/ml of basic fibroblast growth factor (bFGF) resulted in a significant increase in formation of mineralized tissue. The present study was aimed at assessing the effect of bFGF on the proliferation and differentiation of SBMC and on the sequential development of mineralized bone-like tissue in culture. Transmission electron microscopy of bFGF-treated cultures demonstrated the development of a multilayered structure resembling mineralized bone tissue consisting of cell layers embedded within a heavy extracellular matrix. The matrix was rich in bundles of collagen fibers associated with extensive mineral deposits consisting of hydroxyapatite as determined by infrared spectrophotometry. The addition of 3 ng/ml of bFGF resulted in significant enhancement of [3H]thymidine and [3H]proline incorporation and protein accumulation by 12-, 2.5-, and 2.5-fold, respectively. bFGF treatment increased cAMP responsiveness, alkaline phosphatase activity, osteocalcin level, 45Ca2+ deposition, and mineralized-like tissue formation and induced the earlier expression of these markers in the treated culture. A biphasic sequence of events was observed during the development of mineralized bone-like tissue in bFGF-treated and control cultures. The first phase is characterized by cell proliferation and matrix accumulation and is reflected by a progressive increase in [3H]thymidine and [3H]proline incorporation until day 11. The second phase, which follows, is characterized by a sharp decline in cell proliferation and matrix accumulation and a concomitant expression of osteoblast differentiation as reflected by the progressive increase in alkaline phosphatase activity, mineral deposition, and osteocalcin expression. Treatment of cultures with bFGF accentuated this biphasic sequence of events. These results indicate that bFGF has the capacity to stimulate both the growth and the biochemical functions of SBMC obtained from a young adult animal.

Role of apolipoproteins A-I, A-II and C-I in cholesterol efflux from endothelial and smooth muscle cells.

The role of purified apolipoproteins A-I, A-II and C-I in the process of cholesterol efflux from cultured vascular endothelial and smooth muscle cells was studied. [3H]Cholesterol pre-labelled cultures were exposed to serum-free medium supplemented with free apolipoproteins or with apolipoproteins and phosphatidylcholine liposomes and the cholesterol efflux from the cells was determined. Free apolipoproteins A-I and A-II supported cholesterol efflux from vascular endothelial and smooth muscle cells to a higher extent than apolipoprotein C-I. The ability of free apolipoproteins A-I and A-II to support cholesterol efflux was in correlation with their specific binding to the cultures, while no specific binding of apolipoprotein C-I was detected. The association of apolipoprotein A-I with phosphatidylcholine liposomes resulted in a more than two-fold increase in cholesterol efflux compared to free apolipoprotein A-I, while association of apolipoproteins A-II and C-I with phosphatidylcholine liposomes resulted in a very limited increase in cholesterol efflux above that achieved by the free apolipoproteins. These results suggest that apolipoprotein A-I is involved in cholesterol efflux performed by high density lipoprotein. Furthermore, free apolipoproteins A-I and A-II, but not apolipoprotein C-I may take an active part in cholesterol efflux from endothelial and smooth muscle cells.

Metabolism of cholesterol and phospholipids in cultured human vascular smooth muscle cells: differences between artery and vein-derived cells and the effect of oxygen partial pressure.

Human smooth muscle (SM) cells derived from vena saphena magna, aorta abdominalis and arteria mamaria were grown in culture under 40 or 145 mmHg oxygen partial pressure (pO2) and their lipid metabolism studied. Esterification of the cellular [3H]cholesterol was higher by 2.5-fold in artery derived than in vein-derived cells and was slightly higher in cultures exposed to 145 mmHg than to 40 mmHg pO2. Cholesterol efflux in the presence of high density lipoprotein (HDL) in the incubation medium was higher in artery-derived than vein-derived cells. Apolipoprotein (apo) AI also supported cholesterol efflux to a higher extent in artery than in vein-derived cells. Cholesterol efflux in the presence of apo AI was accompanied by a decrease of 50% in cellular [3H]cholesteryl ester in both cell types. SM cultures exposed to [3H]choline incorporated about 90% of the radioactivity to phosphatidylcholine (PC) and 10% to sphingomyelin (SPM). During 5 days exposure to [3H]choline, 10 to 15% and 20 to 30% of the newly synthesized PC and SPM, respectively, were released by vein-derived cells into the incubation medium. The relative amount of SPM of the total radioactive phospholipids released by vein-derived cultures was significantly higher in cultures growing under 40 mmHg than 145 mmHg pO2 reaching a value of up to 33% of the radioactive phospholipids in the incubation medium. HDL was shown to serve as an acceptor for phospholipids released by both vein and artery-derived SM cells, while free apo AI supported phospholipid efflux in artery but not in vein-derived SM cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol efflux from and high-density-lipoproteins binding to cultured bovine vascular endothelial cells are higher than with vascular smooth muscle cells.

Cholesterol metabolism was studied and compared in confluent cultures of adult bovine aortic endothelial and bovine aortic smooth muscle cells which were grown under similar conditions. The total cholesterol content/mg protein was only slightly higher in smooth muscle cells than in endothelial cells and upon exposure to [3H]cholesterol the maximal specific activity/mg protein obtained was similar in both cell types. Most (98%) of the incorporated [3H]cholesterol remained in the form of free cholesterol in both cell types, and provided a system for the study of cholesterol efflux. The role of high-density lipoproteins (HDL) and human serum in cholesterol influx and efflux, in both endothelial and smooth muscle cells, was studied. Net cholesterol transport in the cultures was calculated and net efflux was observed in both cell types. This was higher in endothelial than in smooth muscle cells and HDL was more efficient than human serum in promoting net cholesterol efflux. During the influx experiments, no conversion of [3H]cholesterol to [3H]cholesteryl ester was observed either in the cell layer or in the incubation medium. On the other hand, during efflux experiments when HDL but not human serum was the acceptor, some (about 6%) conversion of [3H]cholesterol to [3H]cholesteryl ester occurred in the incubation medium. 125I-HDL3 binding to endothelial and smooth muscle cells was studied and demonstrated saturation at a concentration of about 100 micrograms protein/ml for both cell types. However, endothelial cells bound about six times more 125I-HDL3 than smooth muscle cells. These studies indicate that vascular endothelial cells are more protected against cholesterol accumulation than vascular smooth muscle cells. The greater ability of endothelial cells to bind HDL complexes when compared with smooth muscle cells, and thereby to be more susceptible to HDL induced cholesterol efflux, may add a new mechanism through which endothelial cells protect the blood vessel from cholesterol accumulation.

Estrogen receptor binding material in blood of patients after clomiphene citrate administration: determination by a radioreceptor assay.

The aim of the current study was to assess whether clomiphene citrate (CC) and/or active metabolites are present at presumed time of ovulation, nidation, or steroid-sensitive organogenesis, in serum of patients receiving CC for induction of ovulation. A radioreceptor assay, based on competitive replacement of 3H-estradiol on the rat uterus estrogen receptor, by ligands present in serum of patients after CC administration, was developed. Ligands reached maximal concentration 4 to 5 hours after a single dose of CC was administered, and declined with a half-life of 4.5 to 10 hours. In patients receiving CC on day 5 to day 9 in the cycle, ligands are still present on day 14 in the cycle and in some patients on day 22 of the cycle, but no ligands were detected 60 days after CC treatment.

Drugs used in ovulation induction. Safety of patient and offspring.

Data on the safety of agents used to induce ovulation in women are presented, with a special reference to the normality of children at birth and puberty. No overall increase in anomalies has been found after the use of clomiphene citrate. This drug has been given up to day 35 of pregnancy, and its safety in these circumstances requires further analysis. More studies will also be needed on offspring as they pass through puberty, to ensure there are no defects in their Müllerian system. No increase in anomalies has been noted after the use of human menopausal gonadotrophins (HMG). The onset of menstrual rhythms and other parameters in the offspring appear to be normal. No increase in various cancers has been identified following the use of clomiphene or HMG, although most treated women have yet to enter the high risk years.